Polynucleotide encoding a novel metalloprotease highly expressed in the testis, MMP-29

ABSTRACT

The present invention provides novel polynucleotides encoding MMP-29 polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel MMP-29 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

This application claims benefit to provisional application U.S. Serial No. 60/286,764 filed Apr. 26, 2001. The entire teachings of the referenced applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides novel polynucleotides encoding MMP-29 polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel MMP-29 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

BACKGROUND OF THE INVENTION

Proteases hydrolyze specific peptide bonds in proteins. The residues at the active site are used to classify proteases (Rawlings & Barrett, 1995). Proteases that hydrolyze peptide bonds using metal ions are referred to as metalloproteases (“MP”). The metalloproteinases may be one of the older classes of proteases and are found in bacteria, fungi as well as in higher organisms. They differ widely in their sequences and their structures, but many contain a zinc ion. In some cases, zinc may be replaced by another metal such as cobalt or nickel.

The gene and protein of the present invention codes for a human protease belonging to the peptidase M10 family (see Rawlings & Barrett, 1995 for review of protease familial classification). This family contains the sequence . . . HE[ILF]GHXXGLXH . . . (SEQ ID NO:81), which is thought to contain amino acids (histidines and or glutamic acid) which coordinate metal ion binding. Such metal ion coordination facilitates catalysis through the stabilization of a noncovalent, tetrahedral intermediate after the attack of a metal-bound water molecule on the carbonyl group of the scissile bond. This intermediate is further decomposed by transfer of the glutamic acid proton to the leaving group. Metal ion coordination is thought to stabilize the negative charges formed within the active site of the enzyme during catalysis. Such stabilization lowers the transition state energy requirements, and thus results in significant rate enhancements during enzymatic catalysis over non-metal ion coordination conditions (Fersht, A., “Enzyme Structure and Mechanism”, 2^(nd) edition, W.H. Freeman and Company, New York, 1985).

Another important feature of peptidase M10 family is a highly conserved octapeptide ( . . . PRC[GN]XP[DR][LIVSAPKQ] . . . (SEQ ID NO:82)) that has been shown to be involved in autoinhibition of metalloproteases (Breathnach R. et al, 1988; Navre M. et al. 1991). A cysteine within the octapeptide chelates the active site zinc ion, thus inhibiting the enzyme. Between the autoinhibitive octapeptide and the catalytic peptide resides a pair of conserved basic amino acids [RK][RK] that serves as the putative cleavage site for activation by furin proteinases.

The prototype of this family is a human secreted interstitial collagenase called matrix metalloproteinase 1. Substrate proteins for the matrix metalloproteinase 1 include the interstitial collagen group—types I, II, III and alpha-macroglobulins (Vincenti MP et al, 1996). A metalloproteinase gene (XMMP) transiently expressed in Xenopus laevis early embryo development has been discovered (Yang M, Murray M T, Kurkinen M, 1997). It is undetected in the blastula stage embryo, induced in gastrula embryo, expressed in neurula embryo, and then down-regulated in pretailbud embryo, suggesting that XMMP plays a role in Xenopus early development. The human MMP-29 gene described herein represents the human ortholog of the Xenopus XMMP.

Metalloproteinases in Disease

Limited-proteolysis by metalloproteases plays a central regulatory role in many physiological and pathophysiological processes. There are many examples of inhibitors of metalloproteases that are useful medications in the treatment of hypertension, heart failure, various forms of cancer and other diseases.

Metalloproteases play many important biological roles in the nervous system, including the spinal cord. There is a balance between the synthesis and degradation of extracellular matrix proteins in the process of synapse formation during development and regeneration. The timing of MP activation is therefore potentially critical. Some MPs have been shown to be upregulated in the spinal cord either during development or in pathological states such as multiple sclerosis, experimental autoimmune encephalomyelitis, and amyotrophic lateral sclerosis. Since MPs degrade extracellular matrix proteins, they would be toxic to developing neurons that depend upon the matrix proteins for survival, neurite outgrowth, and synapse formation. Degradation of the matrix proteins would also cause the breakdown of the blood brain barrier and infiltration of immune cells into the CNS, which occurs in inflammatory conditions such as MS. Other biological processes that metalloproteinases are involved in include fibrillogenesis, angiogenesis, rheumatoid arthritis, osteoarthritis, enamel formation, atherosclerosis, neural degeneration, diabetic renal lesions and ulceration.

Using the above examples, it is clear the availability of a novel cloned metalloproteinase provides opportunities for adjunct or replacement therapy, and are useful for the identification of metalloproteinase agonists, or stimulators (which might stimulate and/or bias metalloproteinase action), as well as, in the identification of metalloproteinase inhibitors. All of which might be therapeutically useful under different circumstances. The metalloproteinase of the present invention can also be used as a scaffold to tailor-make specific metalloproteinase inhibitors.

The inventors of the present invention describe herein, the polynucleotides corresponding to the full-length MMP-29 gene and its encoded polypeptide. Also provided are polypeptide alignments illustrating the strong conservation of the MMP-29 polypeptide to other known metalloproteinases. Data is also provided illustrating the unique tissue expression profile of the MMP-29 polypeptide in testis tissues, which has not been appreciated heretofore.

The invention also provides methods for designing, evaluating and identifying compounds which bind to all or parts of the aforementioned regions. The methods include three dimensional model building (homology modeling) and methods of computer assisted-drug design which can be used to identify compounds which bind or modulate the forementioned regions of the MMP-29 polypeptide. Such compounds are potential inhibitors of MMP-29 or its homologues. The invention also provides novel classes of compounds, and pharmaceutical compositions thereof, that are useful as inhibitors of MMP-29 or its homologues.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of MMP-29 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the MMP-29 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the MMP-29 protein having the amino acid sequence shown in FIGS. 1A–B (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone, MMP-29 (also referred to as BGS26).

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of MMP-29 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the MMP-29 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.

The invention further provides an isolated MMP-29 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

The invention further relates to a polynucleotide encoding a polypeptide fragment of SEQ ID NO:2, or a polypeptide fragment encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.

The invention further relates to a polynucleotide encoding a polypeptide domain of SEQ ID NO:2 or a polypeptide domain encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.

The invention further relates to a polynucleotide encoding a polypeptide epitope of SEQ ID NO:2 or a polypeptide epitope encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.

The invention further relates to a polynucleotide encoding a polypeptide of SEQ ID NO:2 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1, having biological activity.

The invention further relates to a polynucleotide which is a variant of SEQ ID NO:1.

The invention further relates to a polynucleotide which is an allelic variant of SEQ ID NO:1.

The invention further relates to a polynucleotide which encodes a species homologue of the SEQ ID NO:2.

The invention further relates to a polynucleotide which represents the complimentary sequence (antisense) of SEQ ID NO:1.

The invention further relates to a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified herein, wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.

The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:2, wherein the polynucleotide fragment comprises a nucleotide sequence encoding an metalloprotease protein.

The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:2 or the polypeptide encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.

The invention further relates to an isolated nucleic acid molecule of of SEQ ID NO:1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:1 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.

The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:1, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.

The invention further relates to an isolated polypeptide comprising an amino acid sequence that comprises a polypeptide fragment of SEQ ID NO:2 or the encoded sequence included in the deposited clone.

The invention further relates to a polypeptide fragment of SEQ ID NO:2 or the encoded sequence included in the deposited clone, having biological activity.

The invention further relates to a polypeptide domain of SEQ ID NO:2 or the encoded sequence included in the deposited clone.

The invention further relates to a polypeptide epitope of SEQ ID NO:2 or the encoded sequence included in the deposited clone.

The invention further relates to a full length protein of SEQ ID NO:2 or the encoded sequence included in the deposited clone.

The invention further relates to a variant of SEQ ID NO:2.

The invention further relates to an allelic variant of SEQ ID NO:2. The invention further relates to a species homologue of SEQ ID NO:2.

The invention further relates to the isolated polypeptide of of SEQ ID NO:2, wherein the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.

The invention further relates to an isolated antibody that binds specifically to the isolated polypeptide of SEQ ID NO:2.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of SEQ ID NO:2 or the polynucleotide of SEQ ID NO:1.

The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or absence of a mutation in the polynucleotide of SEQ ID NO:1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.

The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or amount of expression of the polypeptide of of SEQ ID NO:2 in a biological sample; and diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.

The invention further relates to a method for identifying a binding partner to the polypeptide of SEQ ID NO:2 comprising the steps of (a) contacting the polypeptide of SEQ ID NO:2 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.

The invention further relates to a gene corresponding to the cDNA sequence of SEQ ID NO:1.

The invention further relates to a method of identifying an activity in a biological assay, wherein the method comprises the steps of expressing SEQ ID NO:1 in a cell, (b) isolating the supernatant; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatant having the activity.

The invention further relates to a process for making polynucleotide sequences encoding gene products having altered SEQ ID NO:2 activity comprising the steps of (a) shuffling a nucleotide sequence of SEQ ID NO:1, (b) expressing the resulting shuffled nucleotide sequences and, (c) selecting for altered activity as compared to the activity of the gene product of said unmodified nucleotide sequence.

The invention further relates to a shuffled polynucleotide sequence produced by a shuffling process, wherein said shuffled DNA molecule encodes a gene product having enhanced tolerance to an inhibitor of SEQ ID NO:2 activity.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is an immune condition.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is reproductive condition.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a female reproductive disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a male reproductive disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is an ovarian disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a testicular disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is an inflammatory disease.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is an inflammatory disease where proteases, preferably metalloproteases, either directly or indirectly, are involved in disease progression.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a cancer.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a gastrointestinal disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a hepatic disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a pulmonary disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a renal disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a cardiovascular disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a neural disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is an immune disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a metabolic disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a skeletal muscle disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a sclerosis.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is amyotrophic lateral sclerosis.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is the juvenile form of amyotrophic lateral sclerosis.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a disorder associated with aberrations of chromosome 2q32.

The invention further relates to a method of identifying a compound that modulates the biological activity of MMP-29, comprising the steps of, (a) combining a candidate modulator compound with MMP-29 having the sequence set forth in one or more of SEQ ID NO:2; and measuring an effect of the candidate modulator compound on the activity of MMP-29.

The invention further relates to a method of identifying a compound that modulates the biological activity of a metalloproteinase, comprising the steps of, (a) combining a candidate modulator compound with a host cell expressing MMP-29 having the sequence as set forth in SEQ ID NO:2; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed MMP-29.

The invention further relates to a method of identifying a compound that modulates the biological activity of MMP-29, comprising the steps of, (a) combining a candidate modulator compound with a host cell containing a vector described herein, wherein MMP-29 is expressed by the cell; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed MMP-29.

The invention further relates to a method of screening for a compound that is capable of modulating the biological activity of MMP-29, comprising the steps of: (a) providing a host cell described herein; (b) determining the biological activity of MMP-29 in the absence of a modulator compound; (c) contacting the cell with the modulator compound; and (d)determining the biological activity of MMP-29 in the presence of the modulator compound; wherein a difference between the activity of MMP-29 in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.

The invention further relates to a compound that modulates the biological activity of human MMP-29 as identified by the methods described herein.

The invention further relates to a computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises the structural coordinates of a member of the group consisting of: the MMP-29 propeptide domain model provided in FIG. 7 in accordance with Table IV; the MMP-29 catalytic domain model provided in FIG. 8 in accordance with Table V; and the MMP-29 hemopexin-like domain model provided in FIG. 9 in accordance with Table VI, wherein said computer comprises: (a) A machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the set of structure coordinates of the model; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central-processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data into said three-dimensional representation; and (d) a display coupled to said central-processing unit for displaying said three-dimensional representation.

The invention further relates to a method for identifying a MMP-29 mutant with altered biological properties, function, or activity using a model selected from the group consisting of: the MMP-29 propeptide domain model provided in FIG. 7 in accordance with Table IV; the MMP-29 catalytic domain model provided in FIG. 8 in accordance with Table V; and the MMP-29 hemopexin-like domain model provided in FIG. 9 in accordance with Table VI, wherein said method comprises the steps of: (a) using a model of said polypeptide according to the structural coordinates of said model to identify amino acids to mutate; and (b) mutating said amino acids to create a mutant MMP-29 protein with altered biological function or properties.

The invention further relates to a method for identifying a MMP-29 mutant with altered biological properties, function, or activity using a model selected from the group consisting of: the MMP-29 propeptide domain model provided in FIG. 7 in accordance with Table IV; the MMP-29 catalytic domain model provided in FIG. 8 in accordance with Table V; and the MMP-29 hemopexin-like domain model provided in FIG. 9 in accordance with Table VI, wherein said method comprises the steps of: (a) using a model of said polypeptide according to the structural coordinates of said model to identify amino acids to mutate; and (b) mutating said amino acids to create a mutant MMP-29 protein with altered biological function or properties wherein the mutant is a member of the group consisting of: (a) a mutant with one or more mutations in the propeptide domain of MMP-29 comprised of amino acids from about A48 to about P120 of SEQ ID NO:2 according to Table IV with altered metalloprotease function or properties; (b) a mutant with one or more mutations in the catalytic domain of MMP-29 comprised of amino acids from about G161 to about E330 of SEQ ID NO:2 according to Table V with altered metalloprotease function or properties; (c) a mutant with one or more mutations in the hemopexin-like domain of MMP-29 comprised of amino acids from about Q346 to about N544 of SEQ ID NO:2 according to Table VI with altered metalloprotease function or properties; (d) a mutant with one or more mutations in the catalytic domain of MMP-29 comprised of amino acids C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and/or Q302 of SEQ ID NO:2 according to Table V with altered metalloprotease function or properties; and (e) a mutant with one or more mutations in the S1 pocket of MMP-29 comprised of amino acids Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2 according to Table V with altered metalloprotease function or properties;;

The invention further relates to a method for designing or selecting compounds as potential modulators of a member of the group consisting of: the MMP-29 propeptide domain model provided in FIG. 7 in accordance with Table IV; the MMP-29 catalytic domain model provided in FIG. 8 in accordance with Table V; and the MMP-29 hemopexin-like domain model provided in FIG. 9 in accordance with Table VI, wherein said method comprises the steps of: (a) identifying a structural or chemical feature of said member using the structural coordinates of said member; (b) and rationally designing compounds that bind to said feature.

The present invention also relates to an isolated polynucleotide consisting of a portion of the human MMP-29 gene consisting of at least 8 bases, specifically excluding Genbank Accession Nos. AW665196, and/or BG234242.

The present invention also relates to an isolated polynucleotide consisting of a nucleotide sequence encoding a fragment of the human MMP-29 protein, wherein said fragment displays one or more functional activities specifically excluding Genbank Accession Nos. AW665196, and/or BG234242.

The present invention also relates to the polynucleotide of SEQ ID NO:1 consisting of at least 10 to 50 bases, wherein said at least 10 to 50 bases specifically exclude the polynucleotide sequence of Genbank Accession Nos. AW665196, and/or BG234242.

The present invention also relates to the polynucleotide of SEQ ID NO:1 consisting of at least 15 to 100 bases, wherein said at least 15 to 100 bases specifically exclude the polynucleotide sequence of Genbank Accession Nos. AW665196, and/or BG234242.

The present invention also relates to the polynucleotide of SEQ ID NO:1 consisting of at least 100 to 1000 bases, wherein said at least 100 to 1000 bases specifically exclude the polynucleotide sequence of Genbank Accession Nos. AW665196, and/or BG234242.

The present invention also relates to an isolated polypeptide fragment of the human MMP-29 protein, wherein said polypeptide fragment does not consist of the polypeptide encoded by the polynucleotide sequence of Genbank Accession Nos. AW665196, and/or BG234242.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIGS. 1A–B show the polynucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) of the novel human metalloproteinase, MMP-29, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence contains a sequence of 1707 nucleotides (SEQ ID NO:1), encoding a polypeptide of 569 amino acids (SEQ ID NO:2). An analysis of the MMP-29 polypeptide determined that it comprised the following features: a predicted signal sequence located from about amino acid 1 to amino acid 24 of SEQ ID NO:2 (FIGS. 1A–B) represented by double underlining; a predicted propeptide domain located from about amino acid 48 to amino acid 120 of SEQ ID NO:2 (FIGS. 1A–B) represented by single underlining; a predicted catalytic domain located from about amino acid 161 to amino acid 330 of SEQ ID NO:2 (FIGS. 1A–B) represented by light shading; a predicted hemopexin-like domain located from about amino acid 346 to amino acid 544 of SEQ ID NO:2 (FIGS. 1A–B) represented by dotted underlinine; three amino acid residues predicted to coordinate the catalytic zinc ion located at amino acid 283, 287, and 293 of SEQ ID NO:2 (FIGS. 1A–B) represented by dark shading; and a predicted “Met-turn” methionine is located at amino acid 301 of SEQ ID NO:2 (FIGS. 1A–B) represented in bold.

FIGS. 2A–C show the regions of identity and similarity between the encoded MMP-29 protein (SEQ ID NO:2) to other metalloproteinases, specifically, the human matrix metalloproteinase 25 protein (MMP-25; SWISS-PROT Accession No: Q9NPA2; SEQ ID NO:7); the human matrix metalloproteinase 17 protein (MMP-17; SWISS-PROT Accession No: Q9ULZ9; SEQ ID NO:5); the rat matrix metalloproteinase 14 protein (MMP-14; SWISS-PROT Accession No: Q10739; SEQ ID NO:6); the Xenopus matrix metalloproteinase protein (XMMP; Genbank Accession No: gi|AAC21447; SEQ ID NO:3); and the Drosophila matrix metalloproteinase 1 protein (Dm1-MMP; Genbank Accession No: gi|AAG33131; SEQ ID NO:4). The alignment was performed using the CLUSTALW algorithm described elsewhere herein. The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Lines between residues indicate gapped regions for the aligned polypeptides.

FIG. 3 shows an expression profile of the novel human metalloproteinase, MMP-29. The figure illustrates the relative expression level of MMP-29 amongst various mRNA tissue sources. As shown, transcripts corresponding to MMP-29 expressed highly in the testis. The MMP-29 polypeptide was also expressed significantly in small intestine, liver, and to a lesser extent, in lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and brain. Expression data was obtained by measuring the steady state MMP-29 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:30 and 31 as described herein.

FIG. 4 shows a hydrophobicity plot of MMP-29 according to the BioPlot Hydrophobicity algorithm of Vector NTI (version 5.5). The six hydrophilic peaks of the HGPRBMY29 polypeptide are clearly visible.

FIG. 5 shows a table illustrating the percent identity and percent similarity between the MMP-29 polypeptide of the present invention with other metalloproteinases, specifically, the human matrix metalloproteinase 25 protein (MMP-25; SWISS-PROT Accession No: Q9NPA2; SEQ ID NO:7); the human matrix metalloproteinase 17 protein (MMP-17; SWISS-PROT Accession No: Q9ULZ9; SEQ ID NO:5); the rat matrix metalloproteinase 14 protein (MMP-14; SWISS-PROT Accession No: Q10739; SEQ ID NO:6); the Xenopus matrix metalloproteinase protein (XMMP; Genbank Accession No: gi|AAC21447; SEQ ID NO:3); and the Drosophila matrix metalloproteinase 1 protein (Dm1-MMP; Genbank Accession No: gi|AAG33131; SEQ ID NO:4). The percent identity and percent similarity values were determined using the GAP algorithm (GCG suite of programs; and Henikoff, S. and Henikoff, J. G., Proc. Natl. Acad. Sci. USA 89: 10915–10919(1992)) using the following parameters: gap weight=8, and length weight=2.

FIG. 6 shows an expanded expression profile of the novel human metalloproteinase, MMP-29. The figure illustrates the relative expression level of MMP-29 amongst various mRNA tissue sources. As shown, the MMP-29 polypeptide was expressed at relatively low levels in a majority of the tissues tested, though predominately in tissues of the female reproductive system, such as the ovary, and significant expression observed in the brain sub region nucleus accumbens, followed by the choroid-plexus and the testis. Expression data was obtained by measuring the steady state MMP-29 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:68 and 69, and Taqman probe (SEQ ID NO:70) as described in Example 5 herein.

FIG. 7 shows a three-dimensional homology model of the propeptide domain of the MMP-29 polypeptide based upon the homologous structure of a portion of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73). The predicted cysteine (“C117”) that is predicted to ligate the catalytic zinc is displayed in space filling representation and labeled, accordingly. The structural coordinates of the propeptide domain of MMP-29 polypeptide are provided in Table IV herein. The homology model of MMP-29 was derived from generating a sequence alignment with the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|942848; SEQ ID NO:73) using the Proceryon suite of software (Proceryon Biosciences, Inc. N.Y., N.Y.), and the overall atomic model including plausible sidechain orientations using the program LOOK (V3.5.2, Molecular Applications Group).

FIGS. 8 shows a three-dimensional homology model of the catalytic domain of the MMP-29 polypeptide based upon the homologous structure of a portion of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73). The predicted active site amino acids of the catalytic domain of the human MMP-29 polypeptide (“H283”, “H287”, and “H293”) are labeled. The structural coordinates of the propeptide domain of MMP-29 polypeptide are provided in Table V herein. The homology model of MMP-29 was derived from generating a sequence alignment with the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) using the Proceryon suite of software (Proceryon Biosciences, Inc. N.Y., N.Y.), and the overall atomic model including plausible sidechain orientations using the program LOOK (V3.5.2, Molecular Applications Group).

FIG. 9 shows a three-dimensional homology model of the hemopexin-like domain of the MMP-29 polypeptide based upon the homologous structure of a portion of the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74). The hemopexin-like domain amino acids of the human MMP-29 polypeptide correspond to amino acids Q346 to N544 of SEQ ID NO:2. The structural coordinates of the hemopexin-like domain of MMP-29 polypeptide are provided in Table VI herein. The homology model of MMP-29 was derived from generating a sequence alignment with the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74) using the Proceryon suite of software (Proceryon Biosciences, Inc. N.Y., N.Y.), and the overall atomic model including plausible sidechain orientations using the program LOOK (V3.5.2, Molecular Applications Group).

FIG. 10 shows an energy graph for the model of the MMP-29 propeptide domain (see FIG. 7) of the present invention (dotted line) and the human fibroblast stromelysin-1 proenzyme template (PDB code 1slmA) (solid line) from which the model was generated. The energy distribution for each protein fold is displayed on the y-axis, while the amino acid residue position of the protein fold is displayed on the x-axis. As shown, the MMP-29 propeptide domain model and 1slmA template have similar energies over the aligned region, suggesting that the structural model of the MMP-29 propeptide domain represents a “native-like” conformation of the MMP-29 propeptide domain. This graph supports the motif and sequence alignments in confirming that the three-dimensional structure coordinates of MMP-29 propeptide domain are an accurate and useful representation of the structure of the MMP-29 propeptide domain.

FIG. 11 shows an energy graph for the model of the MMP-29 catalytic domain (see FIG. 8) of the present invention (dotted line) and the human fibroblast stromelysin-1 proenzyme template (PDB code 1slmA) (solid line) from which the model was generated. The energy distribution for each protein fold is displayed on the y-axis, while the amino acid residue position of the protein fold is displayed on the x-axis. As shown, the MMP-29 catalytic domain model and 1slmA template have similar energies over the aligned region, suggesting that the structural model of the MMP-29 catalytic domain represents a “native-like” conformation of the MMP-29 catalytic domain. This graph supports the motif and sequence alignments in confirming that the three-dimensional structure coordinates of MMP-29 catalytic domain are an accurate and useful representation of the structure of the MMP-29 catalytic domain.

FIG. 12 shows an energy graph for the model of the MMP-29 hemopexin-like domain (see FIG. 9) of the present invention (dotted line) and the pig fibroblast (Interstitial) collagenase Mmp-1 (PDB code 1fblA) (solid line) from which the model was generated. The energy distribution for each protein fold is displayed on the y-axis, while the amino acid residue position of the protein fold is displayed on the x-axis. As shown, the MMP-29 hemopexin-like domain model and 1slmA template have similar energies over the aligned region, suggesting that the structural model of the MMP-29 hemopexin-like domain represents a “native-like” conformation of the MMP-29 hemopexin-like domain. This graph supports the motif and sequence alignments in confirming that the three-dimensional structure coordinates of MMP-29 hemopexin-like domain are an accurate and useful representation of the structure of the MMP-29 hemopexin-like domain.

FIGS. 13A–B shows the regions of identity and similarity between the encoded MMP-29 protein (SEQ ID NO:2) to a portion of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73), and to a portion of the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74). Both stromelysin-1 and collagenase Mmp-1 are metalloproteinases in which the x-ray crystal structure has been solved (Bernstein et al., J. Mol. Biol. 112:535–542 (1977); and Li et al., Structure 3: 541–549 (1995); respectively). As described herein portions of the x-ray structures of the stromelysin-1 proenzyme and collagenase Mmp-1 were used as the basis for building the various homology models of propeptide domain, catalytic domain, and hemopexin-like domain of the MMP-29 polypeptide of the present invention, as represented in Tables IV, V, and VI. Amino acids defining the cysteine switch are highlighted with the (“@”) sign above each intended residue; and the histidines from the catalytic domain are highlighted with asterisks (“*”) above each intended residue.

Table I provides a summary of the novel polypeptides and their encoding polynucleotides of the present invention.

Table II illustrates the preferred hybridization conditions for the polynucleotides of the present invention. Other hybridization conditions may be known in the art or are described elsewhere herein.

Table III provides a summary of various conservative substitutions encompassed by the present invention.

Table IV provides the structural coordinates of the homology model of the propeptide domain of MMP-29 provided in FIG. 7. A description of the headings are as follows: “Atom No” refers to the atom number within the MMP-29 propeptide domain homology model; “Atom Name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid of the MMP-29 polypeptide within which the atom resides, in addition to the amino acid position in which the atom resides; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

Table V provides the structural coordinates of the homology model of the catalytic domain of MMP-29 provided in FIG. 8. A description of the headings are as follows: “Atom No” refers to the atom number within the MMP-29 catalytic domain homology model; “Atom Name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid of the MMP-29 polypeptide within which the atom resides, in addition to the amino acid position in which the atom resides; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

Table VI provides the structural coordinates of the homology model of the hemopexin-like domain of MMP-29 provided in FIG. 9. A description of the headings are as follows: “Atom No” refers to the atom number within the MMP-29 hemopexin-like domain homology model; “Atom Name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid of the MMP-29 polypeptide within which the atom resides, in addition to the amino acid position in which the atom resides; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein.

The invention provides a novel human sequence that encodes a metalloproteinase with substantial homology to the class of metalloproteinases known as matrix metalloproteinases, particularly members of the peptidase M10 class of proteases. Metalloproteinases of this class have been implicated in a number of diseases and disorders which include, for example, fibrillogenesis, angiogenesis, rheumatoid arthritis, osteoarthritis, enamel formation, atherosclerosis, neural degeneration, diabetic renal lesions, and ulceration, for example. In addition, expression analysis indicates the MMP-29 has strong preferential expression in testis; significant expression in small intestine, liver, and to a lesser extent, in lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and brain. Based on this information, we have provisionally named the gene and protein MMP-29.

In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.

In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:1 or the cDNA contained within the clone deposited with the ATCC. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, with or without a signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.

In the present invention, the full length sequence identified as SEQ ID NO:1 was often generated by overlapping sequences contained in one or more clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:1 was deposited with the American Type Culture Collection (“ATCC”). As shown in Table I, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure. The deposited clone is inserted in the pSport1 plasmid (Life Technologies) using the NotI and SalI restriction endonuclease cleavage sites.

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373, preferably a Model 3700, from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

Using the information provided herein, such as the nucleotide sequence in FIGS. 1A–B (SEQ ID NO:1), a nucleic acid molecule of the present invention encoding the MMP-29 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIGS. 1A–B (SEQ ID NO:1) was discovered in a cDNA library derived from human brain and testis.

A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:1, the complement thereof, or the cDNA within the clone deposited with the ATCC. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.

The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1–12 (1983); Seifter et al., Meth Enzymol 182:626–646 (1990); Rattan et al., Ann N.Y. Acad Sci 663:48–62 (1992).)

“SEQ ID NO:1” refers to a polynucleotide sequence while “SEQ ID NO:2” refers to a polypeptide sequence, both sequences identified by an integer specified in Table I.

“A polypeptide having biological activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.)

As used herein the terms “modulate” or “modulates” refer to an increase or decrease in the amount, quality or effect of a particular activity, DNA, RNA, or protein. The definition of “modulate” or “modulates” as used herein is meant to encompass agonists and/or antagonists of a particular activity, DNA, RNA, or protein.

The term “organism” as referred to herein is meant to encompass any organism referenced herein, though preferably to eukaryotic organisms, more preferably to mammals, and most preferably to humans.

The present invention encompasses the identification of proteins, nucleic acids, or other molecules, that bind to polypeptides and polynucleotides of the present invention (for example, in a receptor-ligand interaction). The polynucleotides of the present invention can also be used in interaction trap assays (such as, for example, that described by Ozenberger and Young (Mol Endocrinol., 9(10):1321–9, (1995); and Ann. N.Y. Acad. Sci., 7;766:279–81, (1995)).

The polynucleotide and polypeptides of the present invention are useful as probes for the identification and isolation of full-length cDNAs and/or genomic DNA which correspond to the polynucleotides of the present invention, as probes to hybridize and discover novel, related DNA sequences, as probes for positional cloning of this or a related sequence, as probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides, as probes to quantify gene expression, and as probes for microarrays.

In addition, polynucleotides and polypeptides of the present invention may comprise one, two, three, four, five, six, seven, eight, or more membrane domains.

Also, in preferred embodiments the present invention provides methods for further refining the biological function of the polynucleotides and/or polypeptides of the present invention.

Specifically, the invention provides methods for using the polynucleotides and polypeptides of the invention to identify orthologs, homologs, paralogs, variants, and/or allelic variants of the invention. Also provided are methods of using the polynucleotides and polypeptides of the invention to identify the entire coding region of the invention, non-coding regions of the invention, regulatory sequences of the invention, and secreted, mature, pro-, prepro-, forms of the invention (as applicable).

In preferred embodiments, the invention provides methods for identifying the glycosylation sites inherent in the polynucleotides and polypeptides of the invention, and the subsequent alteration, deletion, and/or addition of said sites for a number of desirable characteristics which include, but are not limited to, augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.

In further preferred embodiments, methods are provided for evolving the polynucleotides and polypeptides of the present invention using molecular evolution techniques in an effort to create and identify novel variants with desired structural, functional, and/or physical characteristics.

The present invention further provides for other experimental methods and procedures currently available to derive functional assignments. These procedures include but are not limited to spotting of clones on arrays, micro-array technology, PCR based methods (e.g., quantitative PCR), anti-sense methodology, gene knockout experiments, and other procedures that could use sequence information from clones to build a primer or a hybrid partner.

Polynucleotides and Polypeptides of the Invention

Features of the Polypeptide Encoded by Gene No:1

The polypeptide of this gene provided as SEQ ID NO:2 (FIGS. 1A–B), encoded by the polynucleotide sequence according to SEQ ID NO:1 (FIGS. 1A–B), and/or encoded by the polynucleotide contained within the deposited clone, MMP-29, has significant homology at the nucleotide and amino acid level to a number of metalloproteinases, which include, the human matrix metalloproteinase 25 protein (MMP-25; SWISS-PROT Accession No: Q9NPA2; SEQ ID NO:7); the human matrix metalloproteinase 17 protein (MMP-17; SWISS-PROT Accession No: Q9ULZ9; SEQ ID NO:5); the rat matrix metalloproteinase 14 protein (MMP-14; SWISS-PROT Accession No: Q10739; SEQ ID NO:6); the Xenopus matrix metalloproteinase protein (XMMP; Genbank Accession No: gi|AAC21447; SEQ ID NO:3); and the Drosophila matrix metalloproteinase 1 protein (Dm1-MMP; Genbank Accession No: gi|AAG33131; SEQ ID NO:4). An alignment of the MMP-29 polypeptide with these proteins is provided in FIGS. 2A–C. Based upon such strong conservation, the inventors have ascribed the MMP-29 polypeptide as having proteolytic activity, preferably metalloproteinase activity.

The MMP-25 matrix metalloproteinase, also referred to as leukolysin and/or MT6-MMP, is thought to function in the activation of progelatinase A. MMP-25 was found to require calcium and zinc for activity, and is attached to the membrane by a GPI-anchor. MMP-25 was found to be expressed predominantly in leukocytes, lung, spleen, in addition to, colon carcinomas, astrocytomas, and glioblastomas. the precursor of MMP-25 is cleaved by a furin endopeptidase. MMP-25 is thought to be specifically involved in the proteolytic arsenal deployed by leukocytes during inflammatory responses (Cell Res. 9 (4), 291–303 (1999)). More recently, MMP-25 has been shown to be expressed in brain tissues where it facilitates tumor progression through its ability to activate progelatinase A at the membrane of cells from colon carcinomas or brain tumors (Cancer Res. 60 (4), 877–882 (2000)). Addition information related to MMP-25 may be found in reference to the following publication: FEBS Lett. 480 (2–3), 142–146 (2000), which is hereby incorporated herein by reference.

The MMP-17 matrix metalloproteinase, also referred to as MT4-MMP, is also thought to function in the activation of membrane-bound precursors of growth factors or inflammatory mediators, such as tumor necrosis factor-alpha. MMP-17 may also be involved in tumoral processes, although its ability to proteolytically activate progelatinase A is not known. Interestingly, MMP-17 is not able to hydrolyse collagen types I, II, III, IV and V, gelatin, fibronectin, laminin, decorin nor alphal-antitrypsin. MMP-17 is a member of the peptidase m10 family, also referred to as the matrixin family, the members of which are zinc metalloproteases that degrade various components of the extracellular matrix, such as fibrin.

MMP-17 requires calcium and zinc for activity, and is capable of cleaving pro-TNF-alpha at the 74-ALA-|-GLN-75 site. Like MMP-25 above, MMP-17 is attached to the membrane by a GPI-anchor. MMP-17 is primarily expressed in brain, leukocytes, colon, ovary, testis and breast cancer, in addition to, many transformed and non-transformed cell types. Additional information relative to MMP-17 may be found in reference to the following publications: Biol. Chem. 380 (9), 1103–1108 (1999); J. Biol. Chem. . . . 274 (48), 34260–34266 (1999); J. Biol. Chem. . . . 274 (46), 33043–33049 (1999); Cancer Res. 56 (5), 944–949 (1996); and FEBS Lett. 457 (3), 353–356 (1999); which are hereby incorporated herein by reference.

The MMP-14 matrix metalloproteinase, also refered to as MT-MMP, seems to specifically activate pro-gelatinase A (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E. & Seiki, M., Nature (London) 370, 61–65 (1994)), and may thus trigger invasion by tumor cells by activating pro-gelatinase A on the tumor cell surface (Proc. Natl. Acad. Sci. U.S.A. 92 (7), 2730–2734 (1995)). Like other matrix metalloproteinases, MT-MMP requires calcium and zinc for activity. Conversely, MT-MMP is believed to represent a type I membrane protein. MMP-14 is a zinc metalloproteinase and belongs to peptidase family M10A.

The Xenopus XMMP metalloproteinase is believed to play a role in Xenopus early development based upon its transient expression in Xenupus embryos (J. Biol. Chem. . . . 272 (21), 13527–13533 (1997)). Specifically, XMMP mRNA was undetected in the blastula stage embryo, induced in gastrula embryo, expressed in neurula embryo, and then down-regulated in pretailbud embryo.

The Dm1-MMP metalloproteinase is believed to be involved in the extracellular matrix remodeling taking place during the development of the central nervous system in Drosophila (J. Biol. Chem. . . . 275 (46), 35978–35985 (2000)).

The MMP-29 polypeptide was determined to have 33.2% identity and 41.0% similarity with the human matrix metalloproteinase 25 protein (MMP-25; SWISS-PROT Accession No: Q9NPA2; SEQ ID NO:7); to have 33.3% identity and 42.1% similarity with the human matrix metalloproteinase 17 protein (MMP-17; SWISS-PROT Accession No: Q9ULZ9; SEQ ID NO:5); to have 35.5% identity and 44.5% similarity with the rat matrix metalloproteinase 14 protein (MMP-14; SWISS-PROT Accession No: Q10739; SEQ ID NO:6); to have 61.6% identity and 70.4% similarity with the Xenopus matrix metalloproteinase protein (XMMP; Genbank Accession No: gi|AAC21447; SEQ ID NO:3); and to have 35.8% identity and 41.4% similarity with the Drosophila matrix metalloproteinase 1 protein (Dm1-MMP; Genbank Accession No: gi|AAG33131; SEQ ID NO:4); as shown in FIG. 5.

The MMP-29 polypeptide was found to contain the conserved sequence HEIGHVLGLPH (SEQ ID NO:8), fitting the consensus sequence pattern of HE[ILF]GHXXGLXH (SEQ ID NO:81) for all metallopoteinases. MMP-29 also contains an octapeptide PRCGVPDM (SEQ ID NO:9) that fits the highly conserved octapeptide pattern of PRC[GN]XP[DR][LIVSAPKQ] (SEQ ID NO:82) that has been shown to be involved in autoinhibition of metalloproteases (Breathnach R. et al, 1988; Navre M. et al. 1991). Between the autoinhibitive octapeptide and the catalytic peptide there are three pair of RR that possibly serve as the putative cleavage site for activation by furin proteinases. Thus, based upon the sequence and structural homology to known metalloproteases, the novel MMP-29 is believed to represent a novel human secreted metalloprotease.

The MMP-29 polypeptide was determined to comprise a signal sequence from about amino acid 1 to about amino acid 24 of SEQ ID NO:2 (FIGS. 1A–B) according to the SPScan computer algorithm (Genetics Computer Group suite of programs). Based upon the predicted signal peptide cleavage site, the mature MMP-29 polypeptide is expected to be from about amino acid 25 to about amino acid 569 of SEQ ID NO:2 (FIGS. 1A–B). As this determination was based upon the prediction from a computer algorithm, the exact physiological cleavage site may vary, as discussed more particularly herein. In this context, the term “about” should be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 more amino acids in either the N- or C-terminal direction of the above referenced polypeptide. Polynucleotides encoding these polypeptides are also provided.

In addition to the mature polypeptide above, the polynucleotides encoding the mature polypeptide are also encompassed by the present invention. Specifically, from about nucleotide position 73 to about nucleotide position 1707 of SEQ ID NO:1 (FIGS. 1A–B).

As discussed more particularly herein, metalloproteinases are a group of structurally diverse, high molecular weight (400 to 500 amino acids) proteins that have a metal ion within their active site, typically zinc. Despite the structural heterogeneity, metalloproteinases share some well defined structural-functional characteristics, particularly in the active site domain (Zhang, X., Gonnella, N C., Koehn, J., Pathak, N., Ganu, V., Melton, R., Parker, D., Hu, SI., Nam, K Y, J. Mol, Biol., 301(2):513–24, (2000)). Non limiting examples of proteins which are known to belong to the metalloproteinase family of proteins are the following: metalloproteinase 1 thru 26 (MMP-1 to MMP-26); membrane-type 1 matrix metalloproteinase (MT1-MMP); Matrilysin-2; Stromelysin-1, Collagenase-1, ADAMs.

More information relating to metalloproteinases can be found elsewhere herein, or in reference to the following publications: Westerk, J., Kahari, V M, FASEB, J., 13(8):781–92, (1999); Ohtani, H, Pathol, Int., 48(1):1–9, (1998); Stack, M S., Ellerbroek, S M., Fishman, D A, Int, J. Oncol., 12(3):569–76, (1998); Tanaka, S., Hamanishi, C., Kikuchi, H., Fukuda, K, Semin, Arthritis, Rheum., 27(6):392–9, (1998); Yu, A E., Hewitt, R E., Connor, E W., Stetler, Stevenson, W G, Drugs, Aging., 11(3):229–44, (1997).

In preferred embodiments, the MMP-29 polypeptide of the present invention is directed to a polypeptide having structural similarity to metalloproteinases.

Based upon the strong homology to members of the metalloproteinase family, the MMP-29 polypeptide is expected to share at least some biological activity with metalloproteinases, preferably with matrix metalloproteinases, in addition to other metalloproteinases referenced herein and/or otherwise known in the art.

Expression profiling designed to measure the steady state mRNA levels encoding the MMP-29 polypeptide showed predominately high expression levels in expressed highly in the testis. The MMP-29 polypeptide was also expressed significantly in small intestine, liver, and to a lesser extent, in lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and brain (See FIG. 4).

Expanded analysis of MMP-29 expression levels by TaqMan™ quantitative PCR (see FIG. 6) confirmed that the MMP-29 polypeptide is expressed at very low levels compared to the results obtained with SYBR green (FIG. 4). MMP-29 mRNA was expression in the majority of the tissues tested, with slightly greater steady state transcript levels observed in tissues of the female reproductive system, with ovary being the highest. Significant expression was also observed in the brain sub region nucleus accumbens, followed by the choroid-plexus, testis, and to a lesser extent in other tissues as shown. These data suggest that modulators of MMP-29 function may have utility in the treatment of various female reproductive disorders including ovarian cancers. Due to MMP-29 expression in the nucleus accumbens, modulators of MMP-29 may also be useful in the treatment of dysphoria, depression, irritability, and anxiety associated with various drug addictions, especially cocaine.

The analysis of MMP-29 expression in various tumor tissue RNA samples indicates that the steady state levels of MMP-29 are 3.4 fold higher in breast tumors than in matched controls. Considerable literature exists on the role of MMPs and their involvement in breast cancer initiation, invasion and metastasis. Consistent with their role in breast cancer progression, high levels of at least two MMPs (MMP-2 and stromelysin-3) have also been found to correlate with poor prognosis in patients with breast cancer (Duffy M J, Maguire T M, Hill A, McDermott E, O'Higgins N. Breast Cancer Res 2000;2(4):252–7). Because MMPs are apparently involved in breast cancer initiation and dissemination, inhibition of MMP-29 may be of value both in preventing breast cancer and in blocking metastasis of established tumors.

The MMP-29 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating cellular adhesion events, cellular proliferation, and inflammation, in various cells, tissues, and organisms, and particularly in mammalian ovary, testis, small intestine, liver, lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and brain tissue, preferably human tissue. MMP-29 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, may be useful in diagnosing, treating, prognosing, and/or preventing reproductive, gastrointestinal, hepatic, pulmonary, cardiovascular, renal, neural, immune, hematopoietic, metabolic, endocrine, and/or proliferative diseases or disorders.

In preferred embodiments, MMP-29 polynucleotides and polypeptides including agonists and fragments thereof, have uses which include treating, diagnosing, prognosing, ameliorating, and/or preventing the following diseases or disorders: fibrinolysis, susceptibility to infectious diseases (such as, for example, AIDS), emphysema, liver cirrhosis, hepatocellular carcinoma, thrombosis, embolisms, thrombin-mediated vascular injury, microcirculation in severe sepsis, arterial thrombosis, myocardial infarction, unstable angina, stroke, venous thrombosis, pulmonary embolism, angiogenesis, rheumatoid arthritis, osteoarthritis, enamel formation, atherosclerosis, neural degeneration, diabetic renal lesions and ulceration, multiple sclerosis, experimental autoimmune encephalomyelitis, amyotrophic lateral sclerosis, degenerative conditions affecting extracellular matrix proteins, conditions affecting neurite outgrowth, and synapse formation.

The strong homology to human metalloproteinases, combined with the predominate localized expression in testis tissue suggests the MMP-29 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing reproductice diseases and disorders, particularly male reproductice disorders In preferred embodiments, MMP-29 polynucleotides and polypeptides including agonists and fragments thereof, have uses which include treating, diagnosing, prognosing, and/or preventing the following, non-limiting, diseases or disorders of the testis: spermatogenesis, infertility, Klinefelter's syndrome, XX male, epididymitis, genital warts, germinal cell aplasia, cryptorchidism, varicocele, immotile cilia syndrome, and viral orchitis. The MMP-29 polynucleotides and polypeptides including agonists and fragments thereof, may also have uses related to modulating testicular development, embryogenesis, reproduction, and in ameliorating, treating, and/or preventing testicular proliferative disorders (e.g., cancers, which include, for example, choriocarcinoma, Nonseminoma, seminona, and testicular germ cell tumors).

Likewise, the predominate localized expression in testis tissue also emphasizes the potential utility for MMP-29 polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing metabolic diseases and disorders which include the following, not limiting examples: premature puberty, incomplete puberty, Kallman syndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis, congenital adrenal hyperplasia, FSH deficiency, and granulomatous disease, for example.

This gene product may also be useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. The testes are also a site of active gene expression of transcripts that is expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications.

Other metalloproteinases are known to be expressed in testis and are believed to play a role in reproductive processes. Specifically, MMP-23 (Velasco, G., Pendas, A, M., Fueyo, A., Knauper, V., Murphy, G., Lopez, Otin, C, J. Biol, Chem., 274(8):4570–6, (1999)), MT4-MMP (Puente, X, S., Pendas, A, M., Llano, E., Velasco, G., Lopez, Otin, C, Cancer, Res., 56(5):944–9, (1996)), MMP-18 (Cossins, J., Dudgeon, T, J., Catlin, G., Gearing, A, J., Clements, J. M, Biochem, Biophys, Res, Commun., 228(2):494–8, (1996)), (Will, H., Hinzmann, B, Eur, J. Biochem., 231(3):602–8, (1995)), ADAM20 and ADAM21 (Poindexter, K., Nelson, N., DuBose, R, F., Black, R, A., Cerretti, D, P, Gene., 237(1):61–70, (1999)), and ADAM29 and ADAM30 (Cerretti, D, P., DuBose, R, F., Black, R, A., Nelson, N, Biochem, Biophys, Res, Commun., 263(3):810–5, (1999)); which are incorporated by reference in their entirety.

The strong homology to human metalloproteinase proteins, combined with the localized expression in small intestine suggests the MMP-29 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing gastrointesinal diseases and/or disorders, which include, but are not limited to, ulcers, irritable bowel syndrome, inflammatory bowel disease, diarrhea, traveler's diarrhea, drug-related diarrhea polyps, absorption disorders, constipation, diverticulitis, vascular disease of the intestines, intestinal obstruction, intestinal infections, ulcerative colitis, Shigellosis, cholera, Crohn's Disease, amebiasis, enteric fever, Whipple's Disease, peritonitis, intrabdominal abcesses, hereditary hemochromatosis, gastroenteritis, viral gastroenteritis, food poisoning, mesenteric ischemia, mesenteric infarction, in addition to, metabolic diseases and/or disorders.

Moreover, polynucleotides and polypeptides, including fragments and/or antagonists thereof, have uses which include, directly or indirectly, treating, preventing, diagnosing, and/or prognosing susceptibility to the following, non-limiting, gastrointestinal infections: Salmonella infection, E.coli infection, E.coli O157:H7 infection, Shiga Toxin-producing E.coli infection, Campylobacter infection (e.g., Campylobacter fetus, Campylobacter upsaliensis, Campylobacter hyointestinalis, Campylobacter lari, Campylobacter jejuni, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter rectus, Campylobacter curvus, Campylobacter sputorum, etc.), Heliobacter infection (e.g., Heliobacter cinaedi, Heliobacter fennelliae, etc.) Yersinia enterocolitica infection, Vibrio sp. Infection (e.g., Vibrio mimicus, Vibrio parahaemolyticus, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio vulnificus, Vibrio alginolyticus, Vibrio metschnikovii, Vibrio damsela, Vibrio cincinnatiensis, etc.) Aeromonas infection (e.g., Aeromonas hydrophila, Aeromonas sobira, Aeromonas caviae, etc.), Plesiomonas shigelliodes infection, Giardia infection (e.g., Giardia lamblia, etc.), Cryptosporidium infection, Listeria infection, Entamoeba histolytica infection, Rotavirus infection, Norwalk virus infection, Clostridium difficile infection, Clostriudium perfringens infection, Staphylococcus infection, Bacillus infection, in addition to any other gastrointestinal disease and/or disorder implicated by the causative agents listed above or elsewhere herein.

Moreover, the tissue distribution in liver indicates the protein product of this clone would be useful for the detection and treatment of liver disorders and cancers. Representative uses are described in the “Hyperproliferative Disorders”, “Infectious Disease”, and “Binding Activity” sections below, and elsewhere herein. Briefly, the protein can be used for the detection, treatment, and/or prevention of hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells, cirrhosis, hepatic cysts, pyrogenic abscess, amebic abcess, hydatid cyst, cystadenocarcinoma, adenoma, focal nodular hyperplasia, hemangioma, hepatocellulae carcinoma, cholangiocarcinoma, angiosarcoma, granulomatous liver disease, . In addition the expression in fetus would suggest a useful role for the protein product in developmental abnormalities, fetal deficiencies, pre-natal disorders and various would-healing diseases and/or tissue trauma.

Moreover, polynucleotides and polypeptides, including fragments and/or antagonists thereof, have uses which include, directly or indirectly, treating, preventing, diagnosing, and/or prognosing the following, non-limiting, hepatic infections: liver disease caused by sepsis infection, liver disease caused by bacteremia, liver disease caused by Pneomococcal pneumonia infection, liver disease caused by Toxic shock syndrome, liver disease caused by Listeriosis, liver disease caused by Legionnaries' disease, liver disease caused by Brucellosis infection, liver disease caused by Neisseria gonorrhoeae infection, liver disease caused by Yersinia infection, liver disease caused by Salmonellosis, liver disease caused by Nocardiosis, liver disease caused by Spirochete infection, liver disease caused by Treponema pallidum infection, liver disease caused by Brrelia burgdorferi infection, liver disease caused by Leptospirosis, liver disease caused by Coxiella burnetii infection, liver disease caused by Rickettsia richettsii infection, liver disease caused by Chlamydia trachomatis infection, liver disease caused by Chlamydia psittaci infection, in addition to any other hepatic disease and/or disorder implicated by the causative agents listed above or elsewhere herein.

In addition, antagonists of the MMP-29 polynucleotides and polypeptides may have uses that include diagnosing, treating, prognosing, and/or preventing diseases or disorders related to hyper metalloproteinase activity, which may include immune and/or proliferative diseases or disorders, particularly thrombosis, embolism, and other blood disorders. Therapeutic and/or pharmaceutical compositions comprising the MMP-29 polypeptides may be formulated to comprise heparin.

Moreover, MMP-29 polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include treating, diagnosing, prognosing, and/or preventing hyperproliferative disorders, particularly of the reproductive, gastrointestinal, and hepatic systems. Such disorders may include, for example, cancers, and metastasis.

MMP-29 polynucleotides and polypeptides, including fragments and/or antagonsists thereof, may have uses which include identification of modulators of MMP-29 function including antibodies (for detection or neutralization), naturally-occurring modulators and small molecule modulators. Antibodies to domains (including MMP-29 epitopes provided herein) of the MMP-29 protein could be used as diagnostic agents of inflammatory conditions in patients, are useful in monitoring the activation and presence of cognate proteases, and can be used as a biomarker for the protease involvement in disease states and in the evaluation of inhibitors of the cognate protease in vivo.

MMP-29 polypeptides and polynucleotides are useful for diagnosing diseases related to over or under expression of MMP-29 proteins by identifying mutations in the MMP-29 gene using MMP-29 probes, or determining MMP-29 protein or mRNA expression levels. MMP-29 polypeptides are also useful for screening for compounds, which affect activity of the protein. Diseases that can be treated with MMP-29 include, the following, non-limiting examples: neuro-regeneration, neuropathic pain, obesity, anorexia, HIV infections, cancers, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, osteoporosis, angina pectoris, myocardial infarction, psychotic, immune, metabolic, cardiovascular, and neurological disorders.

The MMP-29 polynucleotides and polypeptides, including fragments and/or antagonsists thereof, may have used which include identification of modulators of metalloproteinase function including antibodies (for detection or neutralization), naturally-occurring modulators and small molecule modulators. Antibodies to domains of the MMP-29 protein could be used as diagnostic agents of inflammatory conditions in patients, are useful in monitoring the activation and presence of cognate proteases, and can be used as a biomarker for the protease involvement in disease states and in the evaluation of inhibitors of the cognate protease in vivo.

Molecular genetic manipulation of the structure of the active site domain, particularly the metal binding domain, and of other functional domains in the metalloproteinase superfamily enables the production of metalloproteinases with tailor-made activities. Thus, the MMP-29 polypeptides, and fragments thereof, as well as any homologous product resulting from genetic manipulation of the structure, are useful for NMR-based design of modulators of MMP-29 biological activity, and metalloproteinase, in general.

MMP-29 polypeptides and polynucleotides have additional uses which include diagnosing diseases related to the over and/or under expression of MMP-29 by identifying mutations in the MMP-29 gene by using MMP-29 sequences as probes or by determining MMP-29 protein or mRNA expression levels. MMP-29 polypeptides may be useful for screening compounds that affect the activity of the protein. MMP-29 peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with MMP-29 (described elsewhere herein).

Although it is believed the encoded polypeptide may share at least some biological activities with human metalloproteinases (particularly O-sialoglycoprotein endopeptidases), a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the MMP-29 polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from diseased testicular tissue, as compared to, normal tissue might indicate a function in modulating reproductive function, for example. In the case of MMP-29, testis, small intestine, liver, lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and/or brain tissue should be used to extract RNA to prepare the probe.

In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the MMP-29 gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. In the case of MMP-29, a disease correlation related to MMP-29 may be made by comparing the mRNA expression level of MMP-29 in normal tissue, as compared to diseased tissue (particularly diseased tissue isolated from the following: ovary, testis, small intestine, liver, lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and/or brain tissue). Significantly higher or lower levels of MMP-29 expression in the diseased tissue may suggest MMP-29 plays a role in disease progression, and antagonists against MMP-29 polypeptides would be useful therapeutically in treating, preventing, and/or ameliorating the disease. Alternatively, significantly higher or lower levels of MMP-29 expression in the diseased tissue may suggest MMP-29 plays a defensive role against disease progression, and agonists of MMP-29 polypeptides may be useful therapeutically in treating, preventing, and/or ameliorating the disease. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:1 (FIGS. 1A–B).

The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the MMP-29, transforming yeast deficient in metalloproteinase activity, and assessing their ability to grow would provide convincing evidence the MMP-29 polypeptide has metalloproteinase activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.

Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype. Such knock-out experiments are known in the art, some of which are disclosed elsewhere herein.

Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., a testis, small intestine, liver, lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, or brain tissue specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.

In the case of MMP-29 transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (reproductive, gastrointestinal, hepatic, pulmonary, cardiovascular, renal, neural, immune, hematopoietic, metabolic, endocrine, cancers etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.

In preferred embodiments, the following N-terminal MMP-29 deletion polypeptides are encompassed by the present invention: M1-M569, L2-M569, A3-M569, A4-M569, S5-M569, I6-M569, F7-M569, R8-M569, P9-M569, T10-M569, L11-M569, L12-M569, L13-M569, C14-M569, W15-M569, L16-M569, A17-M569, A18-M569, P19-M569, W20-M569, P21-M569, T22-M569, Q23-M569, P24-M569, E25-M569, S26-M569, L27-M569, F28-M569, H29-M569, S30-M569, R31-M569, D32-M569, R33-M569, S34-M569, D35-M569, L36-M569, E37-M569, P38-M569, S39-M569, P40-M569, L41-M569, R42-M569, Q43-M569, A44-M569, K45-M569, P46-M569, I47-M569, A48-M569, D49-M569, L50-M569, H51-M569, A52-M569, A53-M569, Q54-M569, R55-M569, F56-M569, L57-M569, S58-M569, R59-M569, Y60-M569, G61-M569, W62-M569, S63-M569, G64-M569, V65-M569, W66-M569, A67-M569, A68-M569, W69-M569, G70-M569, P71-M569, S72-M569, P73-M569, E74-M569, G75-M569, P76-M569, P77-M569, E78-M569, T79-M569, P80-M569, K81-M569, G82-M569, A83-M569, A84-M569, L85-M569, A86-M569, E87-M569, A88-M569, V89-M569, R90-M569, R91-M569, F92-M569, Q93-M569, R94-M569, A95-M569, N96-M569, A97-M569, L98-M569, P99-M569, A100-M569, S101-M569, G102-M569, E103-M569, L104-M569, D105-M569, A106-M569, A107-M569, T108-M569, L109-M569, A110-M569, A111-M569, M112-M569, N113-M569, R114-M569, P115-M569, R116-M569, C117-M569, G118-M569, V119-M569, P120-M569, D121-M569, M122-M569, R123-M569, P124-M569, P125-M569, P126-M569, P127-M569, S128-M569, A129-M569, P130-M569, P131-M569, S132-M569, P133-M569, P134-M569, G135-M569, P136-M569, P137-M569, P138-M569, R139-M569, A140-M569, R141-M569, S142-M569, R143-M569, R144-M569, S145-M569, P146-M569, R147-M569, A148-M569, P149-M569, L150-M569, S151-M569, L152-M569, S153-M569, R154-M569, R155-M569, G156-M569, W157-M569, Q158-M569, P159-M569, R160-M569, G161-M569, Y162-M569, P163-M569, D164-M569, G165-M569, G166-M569, A167-M569, A168-M569, Q169-M569, A170-M569, F171-M569, S172-M569, K173-M569, R174-M569, T175-M569, L176-M569, S177-M569, W178-M569, R179-M569, L180-M569, L181-M569, G182-M569, E183-M569, A184-M569, L185-M569, S186-M569, S187-M569, Q188-M569, L189-M569, S190-M569, V191-M569, A192-M569, D193-M569, Q194-M569, R195-M569, R196-M569, I197-M569, V198-M569, A199-M569, L200-M569, A201-M569, F202-M569, R203-M569, M204-M569, W205-M569, S206-M569, E207-M569, V208-M569, T209-M569, P210-M569, L211-M569, D212-M569, F213-M569, R214-M569, E215-M569, D216-M569, L217-M569, A218-M569, A219-M569, P220-M569, G221-M569, A222-M569, A223-M569, V224-M569, D225-M569, I226-M569, K227-M569, L228-M569, G229-M569, F230-M569, G231-M569, R232-M569, G233-M569, R234-M569, H235-M569, L236-M569, G237-M569, C238-M569, P239-M569, R240-M569, A241-M569, F242-M569, D243-M569, G244-M569, S245-M569, G246-M569, Q247-M569, E248-M569, F249-M569, A250-M569, H251-M569, A252-M569, W253-M569, R254-M569, L255-M569, G256-M569, D257-M569, I258-M569, H259-M569, F260-M569, D261-M569, D262-M569, D263-M569, E264-M569, H265-M569, F266-M569, T267-M569, P268-M569, P269-M569, T270-M569, S271-M569, D272-M569, T273-M569, G274-M569, I275-M569, S276-M569, L277-M569, L278-M569, K279-M569, V280-M569, A281-M569, V282-M569, H283-M569, E284-M569, I285-M569, G286-M569, H287-M569, V288-M569, L289-M569, G290-M569, L291-M569, P292-M569, H293-M569, T294-M569, Y295-M569, R296-M569, T297-M569, G298-M569, S299-M569, I300-M569, M301-M569, Q302-M569, P303-M569, N304-M569, Y305-M569, I306-M569, P307-M569, Q308-M569, E309-M569, P310-M569, A311-M569, F312-M569, E313-M569, L314-M569, D315-M569, W316-M569, S317-M569, D318-M569, R319-M569, K320-M569, A321-M569, I322-M569, Q323-M569, K324-M569, L325-M569, Y326-M569, G327-M569, S328-M569, C329-M569, E330-M569, G331-M569, S332-M569, F333-M569, D334-M569, T335-M569, A336-M569, F337-M569, D338-M569, W339-M569, I340-M569, R341-M569, K342-M569, E343-M569, R344-M569, N345-M569, Q346-M569, Y347-M569, G348-M569, E349-M569, V350-M569, M351-M569, V352-M569, R353-M569, F354-M569, S355-M569, T356-M569, Y357-M569, F358-M569, F359-M569, R360-M569, N361-M569, S362-M569, W363-M569, Y364-M569, W365-M569, L366-M569, Y367-M569, E368-M569, N369-M569, R370-M569, N371-M569, N372-M569, R373-M569, T374-M569, R375-M569, Y376-M569, G377-M569, D378-M569, P379-M569, I380-M569, Q381-M569, I382-M569, L383-M569, T384-M569, G385-M569, W386-M569, P387-M569, G388-M569, I389-M569, P390-M569, T391-M569, H392-M569, N393-M569, I394-M569, D395-M569, A396-M569, F397-M569, V398-M569, H399-M569, I400-M569, W401-M569, T402-M569, W403-M569, K404-M569, R405-M569, D406-M569, E407-M569, R408-M569, Y409-M569, F410-M569, F411-M569, Q412-M569, G413-M569, N414-M569, Q415-M569, Y416-M569, W417-M569, R418-M569, Y419-M569, D420-M569, S421-M569, D422-M569, K423-M569, D424-M569, Q425-M569, A426-M569, L427-M569, T428-M569, E429-M569, D430-M569, E431-M569, Q432-M569, G433-M569, K434-M569, S435-M569, Y436-M569, P437-M569, K438-M569, L439-M569, I440-M569, S441-M569, E442-M569, G443-M569, F444-M569, P445-M569, G446-M569, I447-M569, P448-M569, S449-M569, P450-M569, L451-M569, D452-M569, T453-M569, A454-M569, F455-M569, Y456-M569, D457-M569, R458-M569, R459-M569, Q460-M569, K461-M569, L462-M569, I463-M569, Y464-M569, F465-M569, F466-M569, K467-M569, E468-M569, S469-M569, L470-M569, V471-M569, F472-M569, A473-M569, F474-M569, D475-M569, V476-M569, N477-M569, R478-M569, N479-M569, R480-M569, V481-M569, L482-M569, N483-M569, S484-M569, Y485-M569, P486-M569, K487-M569, R488-M569, I489-M569, T490-M569, E491-M569, V492-M569, F493-M569, P494-M569, A495-M569, V496-M569, I497-M569, P498-M569, Q499-M569, N500-M569, H501-M569, P502-M569, F503-M569, R504-M569, N505-M569, I506-M569, D507-M569, S508-M569, A509-M569, Y510-M569, Y511-M569, S512-M569, Y513-M569, A514-M569, Y515-M569, N516-M569, S517-M569, I518-M569, F519-M569, F520-M569, F521-M569, K522-M569, G523-M569, N524-M569, A525-M569, Y526-M569, W527-M569, K528-M569, V529-M569, V530-M569, N531-M569, D532-M569, K533-M569, D534-M569, K535-M569, Q536-M569, Q537-M569, N538-M569, S539-M569, W540-M569, L541-M569, P542-M569, A543-M569, N544-M569, G545-M569, L546-M569, F547-M569, P548-M569, K549-M569, K550-M569, F551-M569, I552-M569, S553-M569, E554-M569, K555-M569, W556-M569, F557-M569, D558-M569, V559-M569, C560-M569, D561-M569, V562-M569, and/or H563-M569 of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal MMP-29 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal MMP-29 deletion polypeptides are encompassed by the present invention: M1-M569, M1-N568, M1-L567, M1-T566, M1-S565, M1-I564, M1-H563, M1-V562, M1-D561, M1-C560, M1-V559, M1-D558, M1-F557, M1-W556, M1-K555, M1-E554, M1-S553, M1-I552, M1-F551, M1-K550, M1-K549, M1-P548, M1-F547, M1-L546, M1-G545, M1-N544, M1-A543, M1-P542, M1-L541, M1-W540, M1-S539, M1-N538, M1-Q537, M1-Q536, M1-K535, M1-D534, M1-K533, M1-D532, M1-N531, M1-V530, M1-V529, M1-K528, M1-W527, M1-Y526, M1-A525, M1-N524, M1-G523, M1-K522, M1-F521, M1-F520, M1-F519, M1-I518, M1-S517, M1-N516, M1-Y515, M1-A514, M1-Y513, M1-S512, M1-Y511, M1-Y510, M1-A509, M1-S508, M1-D507, M1-I506, M1-N505, M1-R504, M1-F503, M1-P502, M1-H501, M1-N500, M1-Q499, M1-P498, M1-I497, M1-V496, M1-A495, M1-P494, M1-F493, M1-V492, M1-E491, M1-T490, M1-I489, M1-R488, M1-K487, M1-P486, M1-Y485, M1-S484, M1-N483, M1-L482, M1-V481, M1-R480, M1-N479, M1-R478, M1-N477, M1-V476, M1-D475, M1-F474, M1-A473, M1-F472, M1-V471, M1-L470, M1-S469, M1-E468, M1-K467, M1-F466, M1-F465, M1-Y464, M1-I463, M1-L462, M1-K461, M1-Q460, M1-R459, M1-R458, M1-D457, M1-Y456, M1-F455, M1-A454, M1-T453, M1-D452, M1-L451, M1-P450, M1-S449, M1-P448, M1-I447, M1-G446, M1-P445, M1-F444, M1-G443, M1-E442, M1-S441, M1-I440, M1-L439, M1-K438, M1-P437, Y436, M1-S435, M1-K434, M1-G433, M1-Q432, M1-E431, M1-D430, M1-E429, M1-T428, M1-L427, M1-A426, M1-Q425, M1-D424, M1-K423, M1-D422, M1-S421, M1-D420, M1-Y419, M1-R418, M1-W417, M1-Y416, M1-Q415, M1-N414, M1-G413, M1-Q412, M1-F411, M1-F410, M1-Y409, M1-R408, M1-E407, M1-D406, M1-R405, M1-K404, M1-W403, M1-T402, M1-W401, M1-I400, M1-H399, M1-V398, M1-F397, M1-A396, M1-D395, M1-I394, M1-N393, M1-H392, M1-T391, M1-P390, M1-I389, M1-G388, M1-P387, M1-W386, M1-G385, M1-T384, M1-L383, M1-I382, M1-Q381, M1-I380, M1-P379, M1-D378, M1-G377, M1-Y376, M1-R375, M1-T374, M1-R373, M1-N372, M1-N371, M1-R370, M1-N369, M1-E368, M1-Y367, M1-L366, M1-W365, M1-Y364, M1-W363, M1-S362, M1-N361, M1-R360, M1-F359, M1-F358, M1-Y357, M1-T356, M1-S355, M1-F354, M1-R353, M1-V352, M1-M351, M1-V350, M1-E349, M1-G348, M1-Y347, M1-Q346, M1-N345, M1-R344, M1-E343, M1-K342, M1-R341, M1-I340, M1-W339, M1-D338, M1-F337, M1-A336, M1-T335, M1-D334, M1-F333, M1-S332, M1-G331, M1-E330, M1-C329, M1-S328, M1-G327, M1-Y326, M1-L325, M1-K324, M1-Q323, M1-I322, M1-A321, M1-K320, M1-R319, M1-D318, M1-S317, M1-W316, M1-D315, M1-L314, M1-E313, M1-F312, M1-A311, M1-P310, M1-E309, M1-Q308, M1-P307, M1-I306, M1-Y305, M1-N304, M1-P303, M1-Q302, M1-M301, M1-I300, M1-S299, M1-G298, M1-T297, M1-R296, M1-Y295, M1-T294, M1-H293, M1-P292, M1-L291, M1-G290, M1-L289, M1-V288, M1-H287, M1-G286, M1-I285, M1-E284, M1-H283, M1-V282, M1-A281, M1-V280, M1-K279, M1-L278, M1-L277, M1-S276, M1-I275, M1-G274, M1-T273, M1-D272, M1-S271, M1-T270, M1-P269, M1-P268, M1-T267, M1-F266, M1-H265, M1-E264, M1-D263, M1-D262, M1-D261, M1-F260, M1-H259, M1-I258, M1-D257, M1-G256, M1-L255, M1-R254, M1-W253, M1-A252, M1-H251, M1-A250, M1-F249, M1-E248, M1-Q247, M1-G246, M1-S245, M1-G244, M1-D243, M1-F242, M1-A241, M1-R240, M1-P239, M1-C238, M1-G237, M1-L236, M1-H235, M1-R234, M1-G233, M1-R232, M1-G231, M1-F230, M1-G229, M1-L228, M1-K227, M1-I226, M1-D225, M1-V224, M1-A223, M1-A222, M1-G221, M1-P220, M1-A219, M1-A218, M1-L217, M1-D216, M1-E215, M1-R214, M1-F213, M1-D212, M1-L211, M1-P210, M1-T209, M1-V208, M1-E207, M1-S206, M1-W205, M1-M204, M1-R203, M1-F202, M1-A201, M1-L200, M1-A199, M1-V198, M1-I197, M1-R196, M1-R195, M1-Q194, M1-D193, M1-A192, M1-V191, M1-S190, M1-L189, M1-Q188, M1-S187, M1-S186, M1-L185, M1-A184, M1-E183, M1-G182, M1-L181, M1-L180, M1-R179, M1-W178, M1-S177, M1-L176, M1-T175, M1-R174, M1-K173, M1-S172, M1-F171, M1-A170, M1-Q169, M1-A168, M1-A167, M1-G166, M1-G165, M1-D164, M1-P163, M1-Y162, M1-G161, M1-R160, M1-P159, M1-Q158, M1-W157, M1-G156, M1-R155, M1-R154, M1-S153, M1-L152, M1-S151, M1-L150, M1-P149, M1-A148, M1-R147, M1-P146, M1-S145, M1-R144, M1-R143, M1-S142, M1-R141, M1-A140, M1-R139, M1-P138, M1-P137, M1-P136, M1-G135, M1-P134, M1-P133, M1-S132, M1-P131, M1-P130, M1-A129, M1-S128, M1-P127, M1-P126, M1-P125, M1-P124, M1-R123, M1-M122, M1-D121, M1-P120, M1-V119, M1-G118, M1-C117, M1-R116, M1-P115, M1-R114, M1-N113, M1-M112, M1-A111, M1-A110, M1-L109, M1-T108, M1-A107, M1-A106, M1-D105, M1-L104, M1-E103, M1-G102, M1-S101, M1-A100, M1-P99, M1-L98, M1-A97, M1-N96, M1-A95, M1-R94, M1-Q93, M1-F92, M1-R91, M1-R90, M1-V89, M1-A88, M1-E87, M1-A86, M1-L85, M1-A84, M1-A83, M1-G82, M1-K81, M1-P80, M1-T79, M1-E78, M1-P77, M1-P76, M1-G75, M1-E74, M1-P73, M1-S72, M1-P71, M1-G70, M1-W69, M1-A68, M1-A67, M1-W66, M1-V65, M1-G64, M1-S63, M1-W62, M1-G61, M1-Y60, M1-R59, M1-S58, M1-L57, M1-F56, M1-R55, M1-Q54, M1-A53, M1-A52, M1-H51, M1-L50, M1-D49, M1-A48, M1-I47, M1-P46, M1-K45, M1-A44, M1-Q43, M1-R42, M1-L41, M1-P40, M1-S39, M1-P38, M1-E37, M1-L36, M1-D35, M1-S34, M1-R33, M1-D32, M1-R31, M1-S30, M1-H29, M1-F28, M1-L27, M1-S26, M1-E25, M1-P24, M1-Q23, M1-T22, M1-P21, M1-W20, M1-P19, M1-A18, M1-A17, M1-L16, M1-W15, M1-C14, M1-L13, M1-L12, M1-L11, M1-T10, M1-P9, M1-R8, and/or M1-F7 of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal MMP-29 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

Alternatively, preferred polypeptides of the present invention may comprise polypeptide sequences corresponding to, for example, internal regions of the MMP-29 polypeptide (e.g., any combination of both N- and C-terminal MMP-29 polypeptide deletions) of SEQ ID NO:2. For example, internal regions could be defined by the equation: amino acid NX to amino acid CX, wherein NX refers to any N-terminal deletion polypeptide amino acid of MMP-29 (SEQ ID NO:2), and where CX refers to any C-terminal deletion polypeptide amino acid of MMP-29 (SEQ ID NO:2). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenic epitopes of the MMP-29 polypeptide.

The MMP-29 polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the MMP-29 polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the MMP-29 polypeptide to associate with other polypeptides, particularly the serine protease substrate for MMP-29, or its ability to modulate serine protease function.

Specifically, the MMP-29 polypeptide was predicted to comprise one tyrosine phosphorylation site using the Motif algorithm (Genetics Computer Group, Inc.). Such sites are phosphorylated at the tyrosine amino acid residue. The consensus pattern for tyrosine phosphorylation sites are as follows: [RK]-x(2)-[DE]-x(3)-Y (SEQ ID NO:83), or [RK]-x(3)-[DE]-x(2(SEQ ID NO:84), where Y represents the phosphorylation site and ‘x’ represents an intervening amino acid residue. Additional information specific to tyrosine phosphorylation sites can be found in Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl. Acad. Sci. U.S.A. 79:973–977(1982); Hunter T., J. Biol. Chem. . . . 257:4843–4848(1982), and Cooper J. A., Esch F. S., Taylor S. S., Hunter T., J. Biol. Chem. . . . 259:7835–7841(1984), which are hereby incorporated herein by reference.

In preferred embodiments, the following tyrosine phosphorylation site polypeptides are encompassed by the present invention: QNHPFRNIDSAYYSYAY (SEQ ID NO:22). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these MMP-29 tyrosine phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The MMP-29 polypeptide was predicted to comprise eleven PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177–184(1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. . . . 260:12492–12499(1985); which are hereby incorporated by reference herein.

In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: EGPPETPKGAALA (SEQ ID NO:10), PPRARSRRSPRAP (SEQ ID NO:11), ARSRRSPRAPLSL (SEQ ID NO:12), APLSLSRRGWQPR (SEQ ID NO:13), AAQAFSKRTLSWR (SEQ ID NO:14), SKRTLSWRLLGEA (SEQ ID NO:15), LGLPHTYRTGSIM (SEQ ID NO:16), FELDWSDRKAIQK (SEQ ID NO:17), FVHIWTWKRDERY (SEQ ID NO:18), YWRYDSDKDQALT (SEQ ID NO:19), and/or PKKFISEKWFDVC (SEQ ID NO:20). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the MMP-29 PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The MMP-29 polypeptide has been shown to comprise one glycosylation site according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.

In preferred embodiments, the following asparagine glycosylation site polypeptide is encompassed by the present invention: YENRNNRTRYGDPI (SEQ ID NO:21). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the MMP-29 asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In confirmation of the MMP-29 polypeptide representing a member of the metalloproteinase family, the MMP-29 polypeptide has been shown to comprise one neutral zinc metallopeptidase zinc-binding site domain according to the Motif algorithm (Genetics Computer Group, Inc.). The majority of zinc-dependent metallopeptidases (with the notable exception of the carboxypeptidases) share a common pattern of primary structure in the part of their sequence involved in the binding of zinc, and can be grouped together as a superfamily, known as the metzincins, on the basis of this sequence similarity. They can be classified into a number of distinct families which are listed below along with the proteases which are currently known to belong to these families. Family M1: Bacterial aminopeptidase N (EC 3.4.11.2) (gene pepN), Mammalian aminopeptidase N (EC 3.4.11.2), Mammalian glutamyl aminopeptidase (EC 3.4.11.7) (aminopeptidase A)—which may play a role in regulating growth and differentiation of early B-lineage cells, Yeast aminopeptidase yscII (gene APE2), Yeast alanine/arginine aminopeptidase (gene AAP1), Yeast hypothetical protein YIL37c., Leukotriene A-4 hydrolase (EC 3.3.2.6)—which is the enzyme is responsible for the hydrolysis of an epoxide moiety of LTA-4 to form LTB-4 (it has been shown that it binds zinc and is capable of peptidase activity); Family M2: Angiotensin-converting enzyme (EC 3.4.15.1) (dipeptidyl carboxypeptidase I) (ACE) the enzyme responsible for hydrolyzing angiotensin I to angiotensin II—There are two forms of ACE: a testis-specific isozyme and a somatic isozyme which has two active centers; Family M3: Thimet oligopeptidase (EC 3.4.24.15)—a mammalian enzyme involved in the cytoplasmic degradation of small peptides, Neurolysin (EC 3.4.24.16) (also known as mitochondrial oligopeptidase M or microsomal endopeptidase), Mitochondrial intermediate peptidase precursor (EC 3.4.24.59) (MIP)—which is involved the second stage of processing of some proteins imported in the mitochondrion, Yeast saccharolysin (EC 3.4.24.37) (proteinase yscD), Escherichia coli and related bacteria dipeptidyl carboxypeptidase (EC 3.4.15.5) (gene dcp), Escherichia coli and related bacteria oligopeptidase A (EC 3.4.24.70) (gene opdA or prlC), Yeast hypothetical protein YKL134c; Family M4: Thermostable thermolysins (EC 3.4.24.27), and related thermolabile neutral proteases (bacillolysins) (EC 3.4.24.28) from various species of Bacillus, Pseudolysin (EC 3.4.24.26) from Pseudomonas aeruginosa (gene lasB), Extracellular elastase from Staphylococcus epidermidis, Extracellular protease prt1 from Erwinia carotovora, Extracellular minor protease smp from Serratia marcescens, Vibriolysin (EC 3.4.24.25) from various species of Vibrio, Protease prtA from Listeria monocytogenes, Extracellular proteinase proA from Legionella pneumophila; Family M5: Mycolysin (EC 3.4.24.31) from Streptomyces cacaoi; Family M6: Immune inhibitor A from Bacillus thuringiensis (gene ina). Ina degrades two classes of insect antibacterial proteins, attacins and cecropins; Family M7: Streptomyces extracellular small neutral proteases; Family M8: Leishmanolysin (EC 3.4.24.36) (surface glycoprotein gp63), a cell surface protease from various species of Leishmania; Family M9: Microbial collagenase (EC 3.4.24.3) from Clostridium perfringens and Vibrio alginolyticus; Family M10A: Serralysin (EC 3.4.24.40), an extracellular metalloprotease from Serratia, Alkaline metalloproteinase from Pseudomonas aeruginosa (gene aprA), Secreted proteases A, B, C and G from Erwinia chrysanthemi, Yeast hypothetical protein YIL108w; Family M10B: Mammalian extracellular matrix metalloproteinases (known as matrixins), MMP-1 (EC 3.4.24.7) (interstitial collagenase), MMP-2 (EC 3.4.24.24) (72 Kd gelatinase), MMP-9 (EC 3.4.24.35) (92 Kd gelatinase), MMP-7 (EC 3.4.24.23) (matrylisin), MMP-8 (EC 3.4.24.34) (neutrophil collagenase), MMP-3 (EC 3.4.24.17) (stromelysin-1), MMP-10 (EC 3.4.24.22) (stromelysin-2), and MMP-11 (stromelysin-3), MMP-12 (EC 3.4.24.65) (macrophage metalloelastase), Sea urchin hatching enzyme (envelysin) (EC 3.4.24.12)—A protease that allows the embryo to digest the protective envelope derived from the egg extracellular matrix, Soybean metalloendoproteinase 1; Family M11: Chlamydomonas reinhardtii gamete lytic enzyme (GLE);Family M12A: Astacin (EC 3.4.24.21), a crayfish endoprotease, Meprin A (EC 3.4.24.18), a mammalian kidney and intestinal brush border metalloendopeptidase, Bone morphogenic protein 1 (BMP-1)—a protein which induces cartilage and bone formation and which expresses metalloendopeptidase activity (Drosophila homologue of BMP-1 is the dorsal-ventral patterning protein tolloid), Blastula protease 10 (BP10) from Paracentrotus lividus and the related protein SpAN from Strongylocentrotus purpuratus, Caenorhabditis elegans hypothetical proteins F42A10.8 and R151.5, Choriolysins L and H (EC 3.4.24.67) (also known as embryonic hatching proteins LCE and HCE) from the fish Oryzias lapides—these proteases participates in the breakdown of the egg envelope, which is derived from the egg extracellular matrix, at the time of hatching; Family M12B: Snake venom metalloproteinases—This subfamily mostly groups proteases that act in hemorrhage. Examples are: adamalysin II (EC 3.4.24.46), atrolysin C/D (EC 3.4.24.42), atrolysin E (EC 3.4.24.44), fibrolase (EC 3.4.24.72), trimerelysin I (EC 3.4.25.52) and II (EC 3.4.25.53), Mouse cell surface antigen MS2; Family M13: Mammalian neprilysin (EC 3.4.24.11) (neutral endopeptidase) (NEP), Endothelin-converting enzyme 1 (EC 3.4.24.71) (ECE-1)—which process the precursor of endothelin to release the active peptide, Kell blood group glycoprotein, a major antigenic protein of erythrocytes, The Kell protein is very probably a zinc endopeptidase, Peptidase O from Lactococcus lactis (gene pepO); Family M27: Clostridial neurotoxins, including tetanus toxin (TeTx) and the various botulinum toxins (BoNT)—these toxins are zinc proteases that block neurotransmitter release by proteolytic cleavage of synaptic proteins such as synaptobrevins, syntaxin and SNAP-25; Family M30: Staphylococcus hyicus neutral metalloprotease; Family M32: Thermostable carboxypeptidase 1 (EC 3.4.17.19) (carboxypeptidase Taq)—an enzyme from Thermus aquaticus which is most active at high temperature; Family M34: Lethal factor (LF) from Bacillus anthracis, one of the three proteins composing the anthrax toxin; Family M35: Deuterolysin (EC 3.4.24.39) from Penicillium citrinum and related proteases from various species of Aspergillus; and Family M36: Extracellular elastinolytic metalloproteinases from Aspergillus.

Based on the tertiary structure of thermolysin, the position of the residues acting as zinc ligands and those involved in the catalytic activity are known. Two of the zinc ligands are histidines which are very close together in the sequence; C-terminal to the first histidine is a glutamic acid residue which acts as a nucleophile and promotes the attack of a water molecule on the carbonyl carbon of the substrate. A consensus sequence for neutral zinc metallopeptidases zinc-binding domains is as follows: [GSTALIVN]-x(2)-H-E-[LIVMFYW]-{DEHRKP}-H-x-[LIVMFYWGSPQ] (SEQ ID NO:85), wherein the two H's are zinc ligands, E is the active site residue, and X represents any amino acid.

Additional information relative to metalloproteinases and neutral zinc metallopeptidases zinc-binding domains may be found by reference to the following publications: Jongeneel C. V., Bouvier J., Bairoch A., FEBS Lett. 242:211–214(1989); Murphy G. J. P., Murphy G., Reynolds J. J., FEBS Left. 289:4–7(1991); Bode W., Grams F., Reinemer P., Gomis-Rueth F.-X., Baumann U., McKay D. B., Stoecker W., Zoology 99:237–246(1996); Rawlings N. D., Barrett A. J., Meth. Enzymol. 248:183–228(1995); Woessner J. Jr., FASEB J. 5:2145–2154(1991); Hite L. A., Fox J. W., Bjarnason J. B., Biol. Chem. Hoppe-Seyler 373:381–385(1992); Montecucco C., Schiavo G., Trends Biochem. Sci. 18:324–327(1993); Niemann H., Blasi J., Jahn R., Trends Cell Biol. 4:179–185(1994); which are hereby incorporaed herein by reference in their entirety.

In preferred embodiments, the following neutral zinc metallopeptidases zinc-binding domain polypeptide is encompassed by the present invention: ISLLKVAVHEIGHVLGLPHT (SEQ ID NO:23). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this neutral zinc metallopeptidases zinc-binding domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also provides a three-dimensional homology model of several MMP-29 polypeptide domains. Specifically, three-dimensional homology models of the MMP-29 propeptide, catalytic, and hemopexin-like domains are provided (see FIGS. 7, 8, and 9) representing amino acids from about A48 to about P120, from about G161 to about E330, and from about Q346 to about N544 of SEQ ID NO:2, respectively.

Protein threading and molecular modeling of MMP29 suggests that there are three functional domains for which three dimensional models can be accurately created. Amino acids from about A48 to about P120 of SEQ ID NO:2 comprise the N-terminal domain known as the propeptide region. This region is similar to the propeptide region of human fibroblast Stromelysin-1 proenzyme Protein DataBank (PDB) code (Bernstein et al. 1977) 1slmA. This region contains the cysteine switch. Amino acids from about D121 to about R160 of SEQ ID NO:2 comprise a proline rich linker region. This region is followed by amino acids from about G161 to about E330 of SEQ ID NO:2 which comprise the catalytic domain. Amino acids from about G331 to about N345 of SEQ ID NO:2 make up a linker region between the catalytic domain and the C-terminal hemopexin-like domain. Amino acids from about Q346 to about N544 of SEQ ID NO:2 comprise the hemopexin-like domain which has a short C-terminal region of unknown function composed of amino acids from about G545 to about M569 of SEQ ID NO:2. Based on sequence, structure and known metalloprotease signature sequences, MMP29 is a novel metaloproteinase.

The three dimensional crystallographic and NMR structures of matrix metalloproteinases (MMPs), also referred to as matrixins, allow for the characterization and functional analyses which can be used to understand the role of MMPs in biology and pathology. The detailed structural analysis for MMP29 allows for the characterization of functional domains and development of three dimensional models for the domains of MMP29.

In most cases the matrixins are synthesized as prepro-enzymes and secreted as inactive pro-enzymes. The primary structure and bioinformatics analyses and structural considerations for matrixins and related metalloproteinase families such as the metzincins (astacins, reprolysins, adamalysins, and serralysins) were used to describe the structural and functional organization of the matrixin metalloproteinases (Massova et al., 1998). The matrixins are organized into three distinctive functional domains. The N-terminal domain is a propeptide domain (consisting of about 80–90 amino acids) which has a conserved sequence motif PRCGxPD (SEQ ID NO:86). The cysteine residue within this motif is known as the “cysteine switch”. This cysteine interacts with the catalytic zinc to maintain latency of the pro-enzyme. This domain consists of a three helices connected by loops and turns.

The second domain is the catalytic domain (consisting of about 170 amino acids). The catalytic domain contains a zinc binding motif (HexxHxxGxxH) (SEQ ID NO:87) and a conserved methionine, which forms a unique “Met-turn” structure. The catalytic domain also contains two zinc ions and at least one calcium ion coordinated to various residues. One of the zinc ions is the catalytic zinc and is in the active site. The other zinc ion and (known as the structural zinc) and a calcium ion are located approximately 12 angstroms from the zinc in the active site. The catalytic zinc is essential for proteolytic activity of the matrixins. Three histidine residues coordinate the catalytic zinc and are conserved in all matrixins and related metalloproteinases. This domain consists of a five beta-stranded sheet and three aipha-helices connected by bridging loops (Bode et al., 1993). The structure can be classified as an alpha/beta three-layer sandwich.

The third conserved domain is the hemopexin-like domain (consisting of about 210 amino acids). This domain is a four bladed beta-propeller where each blade consists of four anti-parallel beta-strands and a alpha-helix (Gomis-Ruth et al., 1996) The hemopexin-like domains show sequence and structural similarity to the plasma protein hemopexin. This domain has been shown to play a functional role in substrate binding and interactions with tissue inhibitors of the metalloproteinases.

A three-dimensional homology model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995). The homology model of the MMP-29 propeptide domain, corresponding to amino acid residues A48 to P120 of SEQ ID NO:2, respectively, was based upon the homologous structure of a portion of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) and is defined by the set of structural coordinates set forth in Table IV herein.

The homology model of MMP29 propeptide domain was derived from the sequence alignment set forth in FIG. 13. The sequence identity of the propeptide domain to the structural template is 26%, but there is significant amino acid homology and predicted structural similarity. The three dimensional model for the propeptide domain comprises amino acids A48 through P120 of SEQ ID NO:2. An overall atomic model including plausible side chain orientations was generated using the program LOOK (Levitt, 1992). The three dimensional model for MMP29 propeptide domain is defined by the set of structure coordinates as set forth in Table IV and is shown in FIG. 7 rendered by backbone secondary structures. The cysteine, C117 is the cysteine that ligates the catalytic zinc is also displayed. The role of the cysteine is to ligate the catalytic zinc to maintain the latency of the pro-enzyme while the propeptide is attached. This cysteine is essential and conserved in all matrixin metalloproteinases.

The homology model of the MMP-29 catalytic domain, corresponding to amino acid residues from about G161 to about E330 of SEQ ID NO:2, respectively, was based upon the homologous structure of a portion of the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74) and is defined by the set of structural coordinates set forth in Table V herein.

The homology model of MMP29 catalytic domain was derived from the sequence alignment set forth in FIG. 13. The sequence identity of the catalytic domain to the structural templates is 39%. There is also significant amino acid homology and predicted structural similarity. The three dimensional model for the catalytic domain comprises amino acids G161 through E330 of SEQ ID NO:2. An overall atomic model including plausible side chain orientations was generated using the program LOOK (Levitt, 1992). The three dimensional model for MMP29 catalytic domain is defined by the set of structure coordinates as set forth in Table V and is shown in FIG. 8 rendered by backbone secondary structures. FIG. 8 also displays the side chains for the three histidines (H283, H287, H293) that are predicted to coordinate the catalytic zinc ion. The histidines are essential and conserved in all matrix metalloproteinases.

The homology model of the MMP-29 hemopexin-like domain, corresponding to amino acid residues from about Q346 to about N544 of SEQ ID NO:2, respectively, was based upon the homologous structure of a portion of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) and is defined by the set of structural coordinates set forth in Table VI herein.

The homology model of MMP29 hemopexin-like domain was derived from the sequence alignment set forth in FIG. 13. The sequence identity of the hemopexin-like domain to the template is 23%, but there is significant amino acid homology and predicted structural similarity. The three dimensional model for the hemopexin-like domain comprises amino acids Q346 through N544 of SEQ ID NO:2. An overall atomic model including plausible side chain orientations was generated using the program LOOK (Levitt, 1992). The three dimensional model for MMP29 hemopexin-like domain is defined by the set of structure coordinates as set forth in Table VI and is shown in FIG. 9 rendered by backbone secondary structures.

A description of the headings in Tables IV, V, and VI are as follows: “Atom No” refers to the atom number within the MMP-29 homology model; “Atom name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid within which the atom resides, and the provided number after the amino acid refers to the amino acid number of the “residue”; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

The MMP-29 propeptide, catalytic, and hemopexin-like domain homology models of the present invention may provide one basis for designing rational stimulators (agonists) and/or inhibitors (antagonists) of one or more of the biological functions of MMP-29, or of MMP-29 mutants having altered specificity (e.g., molecularly evolved MMP-29 polypeptides, engineered site-specific MMP-29 mutants, MMP-29 allelic variants, etc.).

Homology models are not only useful for designing rational agonists and/or antagonists, but are also useful in predicting the function of a particular polypeptide. The functional predictions from homology models are typically more accurate than the functional attributes derived from traditional polypeptide sequence homology alignments (e.g., CLUSTALW), particularly when the three dimensional structure of a related polypeptide is known (e.g., 1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73; and 1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74). The increased prediction accuracy is based upon the fact that homology models approximate the three-dimensional structure of a protein, while homology based alignments only take into account the one dimension polypeptide sequence. Since the function of a particular polypeptide is determined not only by its primary, secondary, and tertiary structure, functional assignments derived solely upon homology alignments using the one dimensional protein sequence may be less reliable. A 3-dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995).

Prior to developing a homology model, those of skill in the art would appreciate that a template of a known protein, or model protein, must first be identified which will be used as a basis for constructing the homology model for the protein of unknown structure (query template). In the case of the MMP-29 polypeptide of the present invention, the model protein templates used in constructing the MMP-29 propeptide, catalytic, and hemopexin-like domain homology models were portions of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) and portions of the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74).

Identifying a template can be accomplished using pairwise alignment of protein sequences using such programs as FASTA (Pearson, et al 1990) and BLAST (Altschul, et al, 1990). In cases where sequence similarity is high (greater than 30%), such pairwise comparison methods may be adequate for identifying an appropriate template. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques may be used. Such techniques, include, for example, protein fold recognition (protein threading; Hendlich, et al, 1990), where the compatibility of a particular polypeptide sequence with the 3-dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential.

A pairwise alignment of the MMP-29 polypeptide of the present invention to the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) and the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74) is provided in FIG. 13.

Following the initial sequence alignment, an optional second step would be to optimally align the query template to the model template by manual manipulation and/or by the incorporation of features specific to the polypeptides (e.g., motifs, secondary structure predictions, and allowed conservations). Preferably, the incorporated features are found within both the model and query template.

The next step could be to identify structurally conserved regions that could be used to construct secondary core structure (Sali, et al, 1995). Loops could be added using knowledge-based techniques, and by performing forcefield calculations (Sali, et al, 1995).

In order to recognize errors in a three-dimensional structure, knowledge based mean fields can be used to judge the quality of protein folds (Sippl 1993). The methods can be used to recognize misfolded structures as well as faulty parts of structural models. The technique generates an energy graph where the energy distribution for a given protein fold is displayed on the y-axis and residue position in the protein fold is displayed on the x-axis. The knowledge based mean fields compose a force field derived from a set of globular protein structures taken as a subset from the Protein Data Bank (Bernstein et. al. 1977). To analyze the quality of a model the energy distribution is plotted and compared to the energy distribution of the template from which the model was generated.

FIG. 10 shows the energy graph for the MMP-29 propeptide model (dotted line) and the 1slmA template (solid line) from which the model was generated. It is clear that the model has slightly higher energies in the C-terminal region while the N-terminal region appears to be “native-like”. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of the MMP-29 propeptide domain are an accurate and useful representation for the polypeptide.

FIG. 11 shows the energy graph for the MMP-29 catalytic model (dotted line) and the 1fblA template (solid line) from which the model was generated. It is clear that the model has slightly higher energies in the C-terminal region while the N-terminal region appears to be “native-like”. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of the MMP-29 catalytic domain are an accurate and useful representation for the polypeptide.

FIG. 12 shows the energy graph for the MMP-29 hemopexin-like model (dotted line) and the 1slmA template (solid line) from which the model was generated. It is clear that the model has slightly higher energies in the C-terminal region while the N-terminal region appears to be “native-like”. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of the MMP-29 hemopexin-like domain are an accurate and useful representation for the polypeptide.

The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model.

In this invention, the propeptide domain homology model of residues A48 to about P120 of MMP-29 (SEQ ID NO:2) was derived from generating a sequence alignment with the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) using the Proceryon suite of software (Proceryon Biosciences, Inc., N.Y., N.Y.).

In this invention, the catalytic domain homology model of residues G161 through E330 of MMP-29 (SEQ ID NO:2) was derived from generating a sequence alignment with the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74) using the Proceryon suite of software (Proceryon Biosciences, Inc., N.Y., N.Y.).

In this invention, the hemopexin-like domain homology model of residues Q346 through N544 of MMP-29 (SEQ ID NO:2) was derived from generating a sequence alignment with the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) using the Proceryon suite of software (Proceryon Biosciences, Inc., N.Y., N.Y.).

The sequence alignment was then used to guide three dimensional model construction whereby the backbone and side chain conformations were constructed using the LOOK suite of software (Molecular Applications Group) and homology modeling module SEGMOD (Levitt, M., 1992).

The skilled artisan would appreciate that a set of structure coordinates for a protein represents a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from the generation of similar homology models using different alignment templates (i.e., other than the the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) and the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74), and/or using different methods in generating the homology model, will likely have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Tables IV, V, and VI could be manipulated by fractionalization of the structure coordinates; integer additions, or integer subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.

Therefore, various computational analyses are necessary to determine whether a template molecule or a portion thereof is sufficiently similar to all or part of a query template (e.g., propeptide, catalytic, or hemopexin-like domains of MMP-29) in order to be considered the same. Such analyses may be carried out in current software applications, such as INSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as described in the User's Guide, or software applications available in the SYBYL software suite (Tripos Inc., St. Louis, Mo.).

Using the superimposition tool in the program SYBYL, comparisons can be made between different structures and different conformations of the same structure. The procedure used in SYBYL to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. The atom equivalency within SYBYL is defined by user input. For the purpose of this invention, we will define equivalent atoms as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in angstroms, is reported by the SYBYL program. For the purpose of the present invention, any homology model of MMP-29 that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than 3.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Tables IV, V, or VI are considered identical. More preferably, the root mean square deviation for the homology models of the MMP-29 propeptide, catalytic, or hemopexin-like domains is less than about 2.0 Å, less than about 1.5 Å, less than about 1.0 Å, less than about 0.9 Å, less than about 0.8 Å, less than about 0.7 Å, less than about 0.6 Å, less than about 0.5 Å, less than about 0.4 Å, less than about 0.3 Å, less than about 0.2 Å, or less than about 0.1 Å.

The homology models of the present invention are useful for the structure-based design of modulators of MMP-29 biological function, as well as mutants with altered biological function and/or specificity.

There is 39% sequence identity between catalytic domain of MMP29 and the catalytic domain of matrixin metalloproteinases used in the composite alignment of the human fibroblast Stromelysin-1 proenzyme Protein DataBank (PDB) code (Bernstein et al. 1977) 1slmA (Becker et al., 1995) and the Fibroblast (Interstitial) Collagenase Mmp-1, from pig, PDB code 1fbl (Li et al., 1995) as shown in FIG. 13.

For MMP29 there are functionally important residues are located in the propeptide domain and the catalytic domain. C117 is part of the cysteine switch. This region is part of a conserved motif (PRCGxPD (SEQ ID NO:86)) that forms a loop on which C117 is presented into the active site of the catalytic domain so that the thiol group of the cysteine can interact with the catalytic zinc rendering the pro-enzyme latent. Upon processing the propeptide domain is removed and the active site of the catalytic domain exposed to solvent for substrate binding. In the homology model of the catalytic domain (Table V) residues proposed to coordinate the catalytic zinc ion include: H283, H287, and H293 of SEQ ID NO:2. These residues are conserved in both of the templates used to model the MMP29 domains and in all other matrixin metalloproteinases (denoted by the asterisks (*) in FIG. 13). The conservation of the amino acids that are required for metal coordination emphasizes the key role metal ions play in generating the nucleophile for catalysis. These residues are located in the active site formed at the interface of the propeptide and catalytic domains. The substrate binding cleft is surface exposed adjacent to the catalytic zinc active site and is shallow. The matrixin metalloproteinases also contain a conserved methionine, which forms a unique “Met-turn”, 1-4 beta-turn structure. The conserved methionine of MMP29 that forms the turn that supports the active site is M236 of SEQ ID NO:2. This is part of the local substructure of the active site region and is adjacent to the S1 pocket of the substrate binding site.

In addition to the residues proposed to coordinate the metal ions, there is an adjacent region in the binding site called the S I pocket that can best be described as a hydrophobic pocket. The hydrophobic pocket is composed of residues Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2. The shape and character of the S1 pocket is responsible for substrate specificity for a given matrixin. The more preferred description of the active site includes residues in the hydrophobic pocket, residues that coordinate the metal ion and additional residues that are involved in hydrogen bonding networks and van der Waal's interactions in the region of the active. The binding site residues include but are not limited to: C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2. These active site residues play critical roles in the mechanism of catalysis and substrate specificity and binding.

In a preferred embodiment of the present invention, the molecule comprises the active site region defined by structure coordinates of MMP29 amino acids described above according to Tables IV and V, or a mutant of said molecule. The active site is defined by residues C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2.

More preferred are molecules comprising all or any part of the active site region (C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2) or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said MMP29 amino acids of not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms.

In accordance with the structural coordinates provided in Table IV and the three dimensional homology model of MMP-29, the MMP-29 polypeptide has been shown to comprise a propeptide domain embodied by the following amino acids: at about amino acid A48 to about amino acid P120 of SEQ ID NO:2 (FIGS. 1A–B). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N- or C-terminal direction of the above referenced amino acids.

Also more preferred are polypeptides comprising all or any part of the MMP-29 propeptide domain, or a mutant or homologue of said polypeptide or molecular complex. By mutant or homologue of the molecule is meant a molecule that has a root mean square deviation from the backbone atoms of said MMP-29 amino acids of not not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms.

In preferred embodiments, the following MMP-29 propeptide domain polypeptide is encompassed by the present invention: ADLHAAQRFLSRYGWSGVWAAWGPSPEGPPETPKGAALAEAVRRFQRANA LPASGELDAATLAAMNRPRCGVP (SEQ ID NO:75). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the MMP-29 propeptide domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses polypeptides comprising at least a portion of the MMP-29 propeptide domain (SEQ ID NO:75). Such polypeptides may correspond, for example, to the N- and/or C-terminal deletions of the methionine aminopeptidase domain.

In preferred embodiments, the following N-terminal MMP-29 propeptide domain deletion polypeptides are encompassed by the present invention: A1-P73, D2-P73, L3-P73, H4-P73, A5-P73, A6-P73, Q7-P73, R8-P73, F9-P73, L10-P73, S11-P73, R12-P73, Y13-P73, G14-P73, W15-P73, S16-P73, G17-P73, V18-P73, W19-P73, A20-P73, A21-P73, W22-P73, G23-P73, P24-P73, S25-P73, P26-P73, E27-P73, G28-P73, P29-P73, P30-P73, E31-P73, T32-P73, P33-P73, K34-P73, G35-P73, A36-P73, A37-P73, L38-P73, A39-P73, E40-P73, A41-P73, V42-P73, R43-P73, R44-P73, F45-P73, Q46-P73, R47-P73, A48-P73, N49-P73, A50-P73, L51-P73, P52-P73, A53-P73, S54-P73, G55-P73, E56-P73, L57-P73, D58-P73, A59-P73, A60-P73, T61-P73, L62-P73, A63-P73, A64-P73, M65-P73, N66-P73, and/or R67-P73 of SEQ ID NO:75. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal MMP-29 propeptide domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal MMP-29 propeptide domain deletion polypeptides are encompassed by the present invention: A1-P73, A1-V72, A1-G71, A1-C70, A1-R69, A1-P68, A1-R67, A1-N66, A1-M65, A1-A64, A1-A63, A1-L62, A1-T61, A1-A60, A1-A59, A1-D58, A1-L57, A1-E56, A1-G55, A1-S54, A1-A53, A1-P52, A1-L51, A1-A50, A1-N49, A1-A48, A1-R47, A1-Q46, A1-F45, A1-R44, A1-R43, A1-V42, A1-A41, A1-E40, A1-A39, A1-L38, A1-A37, A36, A1-G35, A1-K34, A1-P33, A1-T32, A1-E31, A1-P30, A1-P29, A1-G28, A1-E27, A1-P26, A1-S25, A1-P24, A1-G23, A1-W22, A1-A21, A1-A20, A1-W19, A1-V18, A1-G17, A1-S16, A1-W15, A1-G14, A1-Y13, A1-R12, A1-S11, A1-L10, A1-F9, A1-R8, and/or A1-Q7 of SEQ ID NO:75. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal MMP-29 propeptide domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of the MMP-29 propeptide domain (e.g., any combination of both N- and C-terminal MMP-29 propeptide domain deletions) of SEQ ID NO:75. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of the MMP-29 propeptide domain (SEQ ID NO:75), and where CX refers to any C-terminal amino acid position of the MMP-29 propeptide domain (SEQ ID NO:75). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following MMP-29 propeptide domain amino acid substitutions are encompassed by the present invention: wherein A48 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D49 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L50 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein H51 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A52 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A53 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q54 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein R55 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F56 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L57 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S58 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein R59 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein Y60 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G61 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W62 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein S63 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G64 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V65 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein W66 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein A67 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A68 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W69 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein G70 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P71 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein S72 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein P73 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein E74 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G75 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P76 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein P77 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein E78 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T79 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein P80 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein K81 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G82 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A83 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A84 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L85 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A86 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E87 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A88 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V89 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein R90 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein R91 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F92 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q93 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein R94 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein A95 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N96 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein A97 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L98 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P99 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein A100 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S101 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G102 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E103 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L104 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D105 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A106 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A107 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T108 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L109 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A110 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A111 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein M112 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein N113 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R114 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein P115 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein R116 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein C117 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G118 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V119 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; and/or wherein P120 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y of SEQ ID NO:2, in addition to any combination thereof. The present invention also encompasses the use of these MMP-29 propeptide domain amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following MMP-29 propeptide domain conservative amino acid substitutions are encompassed by the present invention: wherein A48 is substituted with either a G, I, L, M, S, T, or V; wherein D49 is substituted with an E; wherein L50 is substituted with either an A, I, or V; wherein H51 is substituted with either a K, or R; wherein A52 is substituted with either a G, I, L, M, S, T, or V; wherein A53 is substituted with either a G, I, L, M, S, T, or V; wherein Q54 is substituted with a N; wherein R55 is substituted with either a K, or H; wherein F56 is substituted with either a W, or Y; wherein L57 is substituted with either an A, I, or V; wherein S58 is substituted with either an A, G, M, or T; wherein R59 is substituted with either a K, or H; wherein Y60 is either an F, or W; wherein G61 is substituted with either an A, M, S, or T; wherein W62 is either an F, or Y; wherein S63 is substituted with either an A, G, M, or T; wherein G64 is substituted with either an A, M, S, or T; wherein V65 is substituted with either an A, I, or L; wherein W66 is either an F, or Y; wherein A67 is substituted with either a G, I, L, M, S, T, or V; wherein A68 is substituted with either a G, I, L, M, S, T, or V; wherein W69 is either an F, or Y; wherein G70 is substituted with either an A, M, S, or T; wherein P71 is a P; wherein S72 is substituted with either an A, G, M, or T; wherein P73 is a P; wherein E74 is substituted with a D; wherein G75 is substituted with either an A, M, S, or T; wherein P76 is a P; wherein P77 is a P; wherein E78 is substituted with a D; wherein T79 is substituted with either an A, G, M, or S; wherein P80 is a P; wherein K81 is substituted with either a R, or H; wherein G82 is substituted with either an A, M, S, or T; wherein A83 is substituted with either a G, I, L, M, S, T, or V; wherein A84 is substituted with either a G, I, L, M, S, T, or V; wherein L85 is substituted with either an A, I, or V; wherein A86 is substituted with either a G, I, L, M, S, T, or V; wherein E87 is substituted with a D; wherein A88 is substituted with either a G, I, L, M, S, T, or V; wherein V89 is substituted with either an A, I, or L; wherein R90 is substituted with either a K, or H; wherein R91 is substituted with either a K, or H; wherein F92 is substituted with either a W, or Y; wherein Q93 is substituted with a N; wherein R94 is substituted with either a K, or H; wherein A95 is substituted with either a G, I, L, M, S, T, or V; wherein N96 is substituted with a Q; wherein A97 is substituted with either a G, I, L, M, S, T, or V; wherein L98 is substituted with either an A, I, or V; wherein P99 is a P; wherein A100 is substituted with either a G, I, L, M, S, T, or V; wherein S101 is substituted with either an A, G, M, or T; wherein G102 is substituted with either an A, M, S, or T; wherein E103 is substituted with a D; wherein L104 is substituted with either an A, I, or V; wherein D105 is substituted with an E; wherein A106 is substituted with either a G, I, L, M, S, T, or V; wherein A107 is substituted with either a G, I, L, M, S, T, or V; wherein T108 is substituted with either an A, G, M, or S; wherein L109 is substituted with either an A, I, or V; wherein A110 is substituted with either a G, I, L, M, S, T, or V; wherein A111 is substituted with either a G, I, L, M, S, T, or V; wherein M112 is substituted with either an A, G, S, or T; wherein N113 is substituted with a Q; wherein R114 is substituted with either a K, or H; wherein P115 is a P; wherein R116 is substituted with either a K, or H; wherein C117 is a C; wherein G118 is substituted with either an A, M, S, or T; wherein V119 is substituted with either an A, I, or L; and/or wherein P120 is a P of SEQ ID NO:2 in addition to any combination thereof. Other suitable substitutions within the MMP-29 propeptide domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these MMP-29 propeptide domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In accordance with the structural coordinates provided in Table V and the three dimensional homology model of MMP-29, the MMP-29 polypeptide has been shown to comprise a catalytic domain embodied by the following amino acids: at about amino acid G161 to about amino acid E330 of SEQ ID NO:2 (FIGS. 1A–B). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N- or C-terminal direction of the above referenced amino acids.

Also more preferred are polypeptides comprising all or any part of the MMP-29 catalytic domain, or a mutant or homologue of said polypeptide or molecular complex. By mutant or homologue of the molecule is meant a molecule that has a root mean square deviation from the backbone atoms of said MMP-29 amino acids of not not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms.

In preferred embodiments, the following MMP-29 catalytic domain polypeptide is encompassed by the present invention: GYPDGGAAQAFSKRTLSWRLLGEALSSQLSVADQRRIVALAFRMWSEVTPLD FREDLAAPGAAVDIKLGFGRGRHLGCPRAFDGSGQEFAHAWRLGDIHFDDDE HFTPPTSDTGISLLKVAVHEIGHVLGLPHTYRTGSIMQPNYIPQEPAFELDWSD RKAIQKLYGSCE (SEQ ID NO:76). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the MMP-29 catalytic domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses polypeptides comprising at least a portion of the MMP-29 catalytic domain (SEQ ID NO:76). Such polypeptides may correspond, for example, to the N- and/or C-terminal deletions of the catalytic domain.

In preferred embodiments, the following N-terminal MMP-29 catalytic domain deletion polypeptides are encompassed by the present invention: G1-E170, Y2-E170, P3-E170, D4-E170, G5-E170, G6-E170, A7-E170, A8-E170, Q9-E170, A10-E170, F11-E170, S12-E170, K13-E170, R14-E170, T15-E170, L16-E170, S17-E170, W18-E170, R19-E170, L20-E170, L21-E170, G22-E170, E23-E170, A24-E170, L25-E170, S26-E170, S27-E170, Q28-E170, L29-E170, S30-E170, V31-E170, A32-E170, D33-E170, Q34-E170, R35-E170, R36-E170, I37-E170, V38-E170, A39-E170, L40-E170, A41-E170, F42-E170, R43-E170, M44-E170, W45-E170, S46-E170, E47-E170, V48-E170, T49-E170, P50-E170, L51-E170, D52-E170, F53-E170, R54-E170, E55-E170, D56-E170, L57-E170, A58-E170, A59-E170, P60-E170, G61-E170, A62-E170, A63-E170, V64-E170, D65-E170, I66-E170, K67-E170, L68-E170, G69-E170, F70-E170, G71-E170, R72-E170, G73-E170, R74-E170, H75-E170, L76-E170, G77-E170, C78-E170, P79-E170, R80-E170, A81-E170, F82-E170, D83-E170, G84-E170, S85-E170, G86-E170, Q87-E170, E88-E170, F89-E170, A90-E170, H91-E170, A92-E170, W93-E170, R94-E170, L95-E170, G96-E170, D97-E170, I98-E170, H99-E170, F100-E170, D101-E170, D102-E170, D103-E170, E104-E170, H105-E170, F106-E170, T107-E170, P108-E170, P109-E170, T110-E170, S111-E170, D112-E170, T113-E170, G114-E170, I115-E170, S116-E170, L117-E170, L118-E170, K119-E170, V120-E170, A121-E170, V122-E170, H123-E170, E124-E170, I125-E170, G126-E170, H127-E170, V128-E170, L129-E170, G130-E170, L131-E170, P132-E170, H133-E170, T134-E170, Y135-E170, R136-E170, T137-E170, G138-E170, S139-E170, I140-E170, M141-E170, Q142-E170, P143-E170, N144-E170, Y145-E170, I146-E170, P147-E170, Q148-E170, E149-E170, P150-E170, A151-E170, F152-E170, E153-E170, L154-E170, D155-E170, W156-E170, S157-E170, D158-E170, R159-E170, K160-E170, A161-E170, I162-E170, Q163-E170, and/or K164-E170 of SEQ ID NO:76. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal MMP-29 catalytic domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal MMP-29 catalytic domain deletion polypeptides are encompassed by the present invention: G1-E170, G1-C169, G1-S168, G1-G167, G1-Y166, G1-L165, G1-K164, G1-Q163, G1-I162, G1-A161, G1-K160, G1-R159, G1-D158, G1-S157, G1-W156, G1-D155, G1-L154, G1-E153, G1-F152, G1-A151, G1-P150, G1-E149, G1-Q148, G1-P147, G1-I146, G1-Y145, G1-N144, G1-P143, G1-Q142, G1-M141, G1-I140, G1-S139, G1-G138, G1-T137, G1-R136, G1-Y135, G1-T134, G1-H133, G1-P132, G1-L131, G1-G130, G1-L129, G1-V128, G1-H127, G1-G126, G1-I125, G1-E124, G1-H123, G1-V122, G1-A121, G1-V120, G1-K119, G1-L118, G1-L117, G1-S116, G1-I115, G1-G114, G1-T113, G1-D112, G1-S111, G1-T110, G1-P109, G1-P108, G1-T107, G1-F106, G1-H105, G1-E104, G1-D103, G1-D102, G1-D101, G1-F100, G1-H99, G1-I98, G1-D97, G1-G96, G1-L95, G1-R94, G1-W93, G1-A92, G1-H91, G1-A90, G1-F89, G1-E88, G1-Q87, G1-G86, G1-S85, G1-G84, G1-D83, G1-F82, G1-A81, G1-R80, G1-P79, G1-C78, G1-G77, G1-L76, G1-H75, G1-R74, G1-G73, G1-R72, G1-G71, G1-F70, G1-G69, G1-L68, G1-K67, G1-I66, G1-D65, G1-V64, G1-A63, G1-A62, G1-G61, G1-P60, G1-A59, G1-A58, G1-L57, G1-D56, G1-E55, G1-R54, G1-F53, G1-D52, G1-L51, G1-P50, G1-T49, G1-V48, G1-E47, G1-S46, G1-W45, G1-M44, G1-R43, G1-F42, G1-A41, G1-L40, G1-A39, G1-V38, G1-137, G1-R36, G1-R35, G1-Q34, G1-D33, G1-A32, G1-V31, G1-S30, G1-L29, G1-Q28, G1-S27, G1-S26, G1-L25, G1-A24, G1-E23, G1-G22, G1-L21, G1-L20, G1-R19, G1-W18, G1-S17, G1-L16, G1-T15, G1-R14, G1-K13, G1-S12, G1-F11, G1-A10, G1-Q9, G1-A8, and/or G1-A7 of SEQ ID NO:76. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal MMP-29 catalytic domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of the MMP-29 catalytic domain (e.g., any combination of both N- and C-terminal MMP-29 catalytic domain deletions) of SEQ ID NO:76. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of the MMP-29 catalytic domain (SEQ ID NO:76), and where CX refers to any C-terminal amino acid position of the MMP-29 catalytic domain (SEQ ID NO:76). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following MMP-29 catalytic domain amino acid substitutions are encompassed by the present invention: wherein G161 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y162 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein P163 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein D164 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G165 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G166 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A167 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A168 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q169 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein A170 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F171 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S172 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein K173 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R174 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T175 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L176 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S177 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein W178 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein R179 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L180 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L181 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G182 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E183 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A184 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L185 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S186 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein S187 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Q188 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein L189 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S190 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein V191 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein A192 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D193 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q194 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein R195 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein R196 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein I197 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V198 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein A199 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L200 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A201 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F202 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R203 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein M204 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein W205 is substituted with either an A, C, D, E, F, G, H, I, K,. L, M, N, P, Q, R, S, T, V, or Y; wherein S206 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein E207 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V208 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein T209 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein P210 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein L211 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D212 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F213 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R214 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein E215 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D216 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L217 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A218 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A219 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P220 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein G221 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A222 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A223 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V224 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein D225 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I226 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K227 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L228 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G229 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F230 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G231 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R232 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein G233 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R234 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein H235 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L236 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G237 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C238 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P239 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein R240 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein A241 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F242 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D243 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G244 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S245 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G246 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q247 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein E248 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F249 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A250 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H251 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A252 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W253 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein R254 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L255 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G256 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D257 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I258 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H259 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F260 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D261 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D262 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D263 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E264 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H265 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F266 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T267 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein P268 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein P269 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein T270 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein S271 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D272 is substituted with either an A, C, E, F, G, H, I, K, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G274 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I275 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S276 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein L277 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L278 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein K279 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V280 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein A281 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V282 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein H283 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E284 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I285 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G286 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H287 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V288 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L289 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G290 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L291 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P292 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein H293 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T294 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein Y295 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein R296 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T297 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G298 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S299 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein I300 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein M301 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein Q302 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein P303 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein N304 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein Y305 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein I306 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P307 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein Q308 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein E309 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P310 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein A311 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F312 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E313 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L314 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D315 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W316 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein S317 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D318 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R319 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein K320 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A321 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I322 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q323 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein K324 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L325 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Y326 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G327 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S328 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein C329 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and/or wherein E330 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y of SEQ ID NO:2, in addition to any combination thereof. The present invention also encompasses the use of these MMP-29 catalytic domain amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following MMP-29 catalytic domain conservative amino acid substitutions are encompassed by the present invention: wherein G161 is substituted with either an A, M, S, or T; wherein Y162 is either an F, or W; wherein P163 is a P; wherein D164 is substituted with an E; wherein G165 is substituted with either an A, M, S, or T; wherein G166 is substituted with either an A, M, S, or T; wherein A167 is substituted with either a G, I, L, M, S, T, or V; wherein A168 is substituted with either a G, I, L, M, S, T, or V; wherein Q169 is substituted with a N; wherein A170 is substituted with either a G, I, L, M, S, T, or V; wherein F171 is substituted with either a W, or Y; wherein S172 is substituted with either an A, G, M, or T; wherein K173 is substituted with either a R, or H; wherein R174 is substituted with either a K, or H; wherein T175 is substituted with either an A, G, M, or S; wherein L176 is substituted with either an A, I, or V; wherein S177 is substituted with either an A, G, M, or T; wherein W178 is either an F, or Y; wherein R179 is substituted with either a K, or H; wherein L180 is substituted with either an A, I, or V; wherein L181 is substituted with either an A, I, or V; wherein G182 is substituted with either an A, M, S, or T; wherein E183 is substituted with a D; wherein A184 is substituted with either a G, I, L, M, S, T, or V; wherein L185 is substituted with either an A, I, or V; wherein S186 is substituted with either an A, G, M, or T; wherein S187 is substituted with either an A, G, M, or T; wherein Q188 is substituted with a N; wherein L189 is substituted with either an A, I, or V; wherein S190 is substituted with either an A, G, M, or T; wherein V191 is substituted with either an A, I, or L; wherein A192 is substituted with either a G, I, L, M, S, T, or V; wherein D193 is substituted with an E; wherein Q194 is substituted with a N; wherein R195 is substituted with either a K, or H; wherein R196 is substituted with either a K, or H; wherein I197 is substituted with either an A, V, or L; wherein V198 is substituted with either an A, I, or L; wherein A199 is substituted with either a G, I, L, M, S, T, or V; wherein L200 is substituted with either an A, I, or V; wherein A201 is substituted with either a G, I, L, M, S, T, or V; wherein F202 is substituted with either a W, or Y; wherein R203 is substituted with either a K, or H; wherein M204 is substituted with either an A, G, S, or T; wherein W205 is either an F, or Y; wherein S206 is substituted with either an A, G, M, or T; wherein E207 is substituted with a D; wherein V208 is substituted with either an A, I, or L; wherein T209 is substituted with either an A, G, M, or S; wherein P210 is a P; wherein L211 is substituted with either an A, I, or V; wherein D212 is substituted with an E; wherein F213 is substituted with either a W, or Y; wherein R214 is substituted with either a K, or H; wherein E215 is substituted with a D; wherein D216 is substituted with an E; wherein L217 is substituted with either an A, I, or V; wherein A218 is substituted with either a G, I, L, M, S, T, or V; wherein A219 is substituted with either a G, I, L, M, S, T, or V; wherein P220 is a P; wherein G221 is substituted with either an A, M, S, or T; wherein A222 is substituted with either a G, I, L, M, S, T, or V; wherein A223 is substituted with either a G, I, L, M, S, T, or V; wherein V224 is substituted with either an A, I, or L; wherein D225 is substituted with an E; wherein I226 is substituted with either an A, V, or L; wherein K227 is substituted with either a R, or H; wherein L228 is substituted with either an A, I, or V; wherein G229 is substituted with either an A, M, S, or T; wherein F230 is substituted with either a W, or Y; wherein G231 is substituted with either an A, M, S, or T; wherein R232 is substituted with either a K, or H; wherein G233 is substituted with either an A, M, S, or T; wherein R234 is substituted with either a K, or H; wherein H235 is substituted with either a K, or R; wherein L236 is substituted with either an A, I, or V; wherein G237 is substituted with either an A, M, S, or T; wherein C238 is a C; wherein P239 is a P; wherein R240 is substituted with either a K, or H; wherein A241 is substituted with either a G, I, L, M, S, T, or V; wherein F242 is substituted with either a W, or Y; wherein D243 is substituted with an E; wherein G244 is substituted with either an A, M, S, or T; wherein S245 is substituted with either an A, G, M, or T; wherein G246 is substituted with either an A, M, S, or T; wherein Q247 is substituted with a N; wherein E248 is substituted with a D; wherein F249 is substituted with either a W, or Y; wherein A250 is substituted with either a G, I, L, M, S, T, or V; wherein H251 is substituted with either a K, or R; wherein A252 is substituted with either a G, I, L, M, S, T, or V; wherein W253 is either an F, or Y; wherein R254 is substituted with either a K, or H; wherein L255 is substituted with either an A, I, or V; wherein G256 is substituted with either an A, M, S, or T; wherein D257 is substituted with an E; wherein I258 is substituted with either an A, V, or L; wherein H259 is substituted with either a K, or R; wherein F260 is substituted with either a W, or Y; wherein D261 is substituted with an E; wherein D262 is substituted with an E; wherein D263 is substituted with an E; wherein E264 is substituted with a D; wherein H265 is substituted with either a K, or R; wherein F266 is substituted with either a W, or Y; wherein T267 is substituted with either an A, G, M, or S; wherein P268 is a P; wherein P269 is a P; wherein T270 is substituted with either an A, G, M, or S; wherein S271 is substituted with either an A, G, M, or T; wherein D272 is substituted with an E; wherein T273 is substituted with either an A, G, M, or S; wherein G274 is substituted with either an A, M, S, or T; wherein I275 is substituted with either an A, V, or L; wherein S276 is substituted with either an A, G, M, or T; wherein L277 is substituted with either an A, I, or V; wherein L278 is substituted with either an A, I, or V; wherein K279 is substituted with either a R, or H; wherein V280 is substituted with either an A, I, or L; wherein A281 is substituted with either a G, I, L, M, S, T, or V; wherein V282 is substituted with either an A, I, or L; wherein H283 is substituted with either a K, or R; wherein E284 is substituted with a D; wherein I285 is substituted with either an A, V, or L; wherein G286 is substituted with either an A, M, S, or T; wherein H287 is substituted with either a K, or R; wherein V288 is substituted with either an A, I, or L; wherein L289 is substituted with either an A, I, or V; wherein G290 is substituted with either an A, M, S, or T; wherein L291 is substituted with either an A, I, or V; wherein P292 is a P; wherein H293 is substituted with either a K, or R; wherein T294 is substituted with either an A, G, M, or S; wherein Y295 is either an F, or W; wherein R296 is substituted with either a K, or H; wherein T297 is substituted with either an A, G, M, or S; wherein G298 is substituted with either an A, M, S, or T; wherein S299 is substituted with either an A, G, M, or T; wherein I300 is substituted with either an A, V, or L; wherein M301 is substituted with either an A, G, S, or T; wherein Q302 is substituted with a N; wherein P303 is a P; wherein N304 is substituted with a Q; wherein Y305 is either an F, or W; wherein I306 is substituted with either an A, V, or L; wherein P307 is a P; wherein Q308 is substituted with a N; wherein E309 is substituted with a D; wherein P310 is a P; wherein A311 is substituted with either a G, I, L, M, S, T, or V; wherein F312 is substituted with either a W, or Y; wherein E313 is substituted with a D; wherein L314 is substituted with either an A, I, or V; wherein D315 is substituted with an E; wherein W316 is either an F, or Y; wherein S317 is substituted with either an A, G, M, or T; wherein D318 is substituted with an E; wherein R319 is substituted with either a K, or H; wherein K320 is substituted with either a R, or H; wherein A321 is substituted with either a G, I, L, M, S, T, or V; wherein I322 is substituted with either an A, V, or L; wherein Q323 is substituted with a N; wherein K324 is substituted with either a R, or H; wherein L325 is substituted with either an A, I, or V; wherein Y326 is either an F, or W; wherein G327 is substituted with either an A, M, S, or T; wherein S328 is substituted with either an A, G, M, or T; wherein C329 is a C; and/or wherein E330 is substituted with a D of SEQ ID NO:2 in addition to any combination thereof. Other suitable substitutions within the MMP-29 catalytic domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these MMP-29 catalytic domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In accordance with the structural coordinates provided in Table VI and the three dimensional homology model of MMP-29, the MMP-29 polypeptide has been shown to comprise a hemopexin-like domain embodied by the following amino acids: at about amino acid Q346 to about amino acid N544 of SEQ ID NO:2 (FIGS. 1A–B). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N- or C-terminal direction of the above referenced amino acids.

Also more preferred are polypeptides comprising all or any part of the MMP-29 hemopexin-like domain, or a mutant or homologue of said polypeptide or molecular complex. By mutant or homologue of the molecule is meant a molecule that has a root mean square deviation from the backbone atoms of said MMP-29 amino acids of not not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0. I Angstroms.

In preferred embodiments, the following MMP-29 hemopexin-like domain polypeptide is encompassed by the present invention: QYGEVMVRFSTYFFRNSWYWLYENRNNRTRYGDPIQILTGWPGIPTHNIDAF VHIWTWKRDERYFFQGNQYWRYDSDKDQALTEDEQGKSYPKLISEGFPGIPS PLDTAFYDRRQKLIYFFKESLVFAFDVNRNRVLNSYPKRITEVFPAVIPQNHPF RNIDSAYYSYAYNSIFFFKGNAYWKVVNDKDKQQNSWLPAN (SEQ ID NO:77). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the MMP-29 hemopexin-like domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses polypeptides comprising at least a portion of the MMP-29 hemopexin-like domain (SEQ ID NO:77). Such polypeptides may correspond, for example, to the N- and/or C-terminal deletions of the hemopexin-like domain.

In preferred embodiments, the following N-terminal MMP-29 hemopexin-like domain deletion polypeptides are encompassed by the present invention: Q1-N199, Y2-N199, G3-N199, E4-N199, V5-N199, M6-N199, V7-N199, R8-N199, F9-N199, S10-N199, T11-N199, Y12-N199, F13-N199, F14-N199, R15-N199, N16-N199, S17-N199, W18-N199, Y19-N199, W20-N199, L21-N199, Y22-N199, E23-N199, N24-N199, R25-N199, N26-N199, N27-N199, R28-N199, T29-N199, R30-N199, Y31-N199, G32-N199, D33-N199, P34-N199, I35-N199, Q36-N199, N199, I37-N199, L38-N199, T39-N199, G40-N199, W41-N199, P42-N199, G43-N199, I44-N199, P45N199, T46-N199, H47-N199, N48-N199, I49-N199, D50-N199, A51-N199, F52-N199, V53-N199, H54-N199, I55-N199, W56-N199, T57-N199, W58-N199, K59-N199, R60-N199, D61-N199, E62-N199, R63-N199, Y64-N199, F65-N199, F66-N199, Q67-N199, G68-N199, N69-N199, Q70-N199, Y71-N199, W72-N199, R73-N199, Y74-N199, D75-N199, S76-N199, D77-N199, K78-N199, D79-N199, Q80-N199, A81-N199, L82-N199, T83-N199, E84-N199, D85-N199, E86-N199, Q87-N199, G88-N199, K89-N199, S90-N199, Y91-N199, P92-N199, K93-N199, L94-N199, I95-N199, S96-N199, E97-N199, G98-N199, F99-N199, P100-N199, G101-N199, I102-N199, P103-N199, S104-N199, P105-N199, L106-N199, D107-N199, T108-N199, A109-N199, F110-N199, Y111-N199, D112-N199, R113-N199, R114-N199, Q115-N199, K116-N199, L117-N199, I118-N199, Y119-N199, F120-N199, F121-N199, K122-N199, E123-N199, S124-N199, L125-N199, V126-N199, F127-N199, A128-N199, F129-N199, D130-N199, V131-N199, N132-N199, R133-N199, N134-N199, R135-N199, V136-N199, L137-N199, N138-N199, S139-N199, Y140-N199, P141-N199, K142-N199, R143-N199, I144-N199, T145-N199, E146-N199, V147-N199, F148-N199, P149-N199, A150-N199, V151-N199, I152-N199, P153-N199, Q154-N199, N155-N199, H156-N199, P157-N199, F158-N199, R159-N199, N160-N199, I161-N199, D162-N199, S163-N199, A164-N199, Y165-N199, Y166-N199, S167-N199, Y168-N199, A169-N199, Y170-N199, N171-N199, S172-N199, I173-N199, F174-N199, F175-N199, F176-N199, K177-N199, G178-N199, N179-N199, A180-N199, Y181-N199, W182-N199, K183-N199, V184-N199, V185-N199, N186-N199, D187-N199, K188-N199, D189-N199, K190-N199, Q191-N199, Q192-N199, and/or N193-N199 of SEQ ID NO:77. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal MMP-29 hemopexin-like domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal MMP-29 hemopexin-like domain deletion polypeptides are encompassed by the present invention: Q1-N199, Q1-A198, Q1-P197, Q1-L196, Q1-W195, Q1-S194, Q1-N193, Q1-Q192, Q1-Q191, Q1-K190, Q1-D189, Q1-K188, Q1-D187, Q1-N186, Q1-V185, Q1-V184, Q1-K183, Q1-W182, Q1-Y181, Q1-A180, Q1-N179, Q1-G178, Q1-K177, Q1-F176, Q1-F175, Q1-F174, Q1-1173, Q1-S172, Q1-N171, Q1-Y170, Q1-A169, Q1-Y168, Q1-S167, Q1-Y166, Q1-Y165, Q1-A164, Q1-S163, Q1-D162, Q1-I161, Q1-N160, Q1-R159, Q1-F158, Q1-P157, Q1-H156, Q1-N155, Q1-Q154, Q1-P153, Q1-I152, Q1-V151, Q1-A150, Q1-P149, Q1-F148, Q1-V147, Q1-E146, Q1-T145, Q1-I144, Q1-R143, Q1-K142, Q1-P141, Q1-Y140, Q1-S139, Q1-N138, Q1-L137, Q1-V136, Q1-R135, Q1-N134, Q1-R133, Q1-N132, Q1-V131, Q1-D130, Q1-F129, Q1-A128, Q1-F127, Q1-V126, Q1-L125, Q1-S124, Q1-E123, Q1-K122, Q1-F121, Q1-F120, Q1-Y119, Q1-I118, Q1-L117, Q1-K116, Q1-Q115, Q1-R114, Q1-R113, Q1-D112, Q1-Y111, Q1-F110, Q1-A109, Q1-T108, Q1-D107, Q1-L106, Q1-P105, Q1-S104, Q1-P103, Q1-I102, Q1-G101, Q1-P100, Q1-F99, Q1-G98, Q1-E97, Q1-S96, Q1-I95, Q1-L94, Q1-K93, Q1-P92, Q1-Y91, Q1-S90, Q1-K89, Q1-G88, Q1-Q87, Q1-E86, Q1-D85, Q1-E84, Q1-T83, Q1-L82, Q1-A81, Q1-Q80, Q1-D79, Q1-K78, Q1-D77, Q1-S76, Q1-D75, Q1-Y74, Q1-R73, Q1-W72, Q1-Y71, Q1-Q70, Q1-N69, Q1-G68, Q1-Q67, Q1-F66, Q1-F65, Q1-Y64, Q1-R63, Q1-E62, Q1-D61, Q1-R60, Q1-K59, Q1-W58, Q1-T57, Q1-W56, Q1-I55, Q1-H54, Q1-V53, Q1-F52, Q1-A51, Q1-D50, Q1-I49, Q1-N48, Q1-H47, Q1-T46, Q1-P45, Q1-I44, Q1-G43, Q1-P42, Q1-W41, Q1-G40, Q1-T39, Q1-L38, Q1-I37, Q1-Q36, Q1-I35, Q1-P34, Q1-D33, Q1-G32, Q1-Y31, Q1-R30, Q1-T29, Q1-R28, Q1-N27, Q1-N26, Q1-R25, Q1-N24, Q1-E23, Q1-Y22, Q1-L21, Q1-W20, Q1-Y19, Q1-W18, Q1-S17, Q1-N16, Q1-R15, Q1-F14, Q1-F13, Q1-Y12, Q1-T11, Q1-S10, Q1-F9, Q1-R8, and/or Q1-V7 of SEQ ID NO:77. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal MMP-29 hemopexin-like domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of the MMP-29 hemopexin-like domain (e.g., any combination of both N- and C-terminal MMP-29 hemopexin-like domain deletions) of SEQ ID NO:77. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of the MMP-29 hemopexin-like domain (SEQ ID NO:77), and where CX refers to any C-terminal amino acid position of the MMP-29 hemopexin-like domain (SEQ ID NO:77). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following MMP-29 hemopexin-like domain amino acid substitutions are encompassed by the present invention: wherein Q346 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein Y347 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G348 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E349 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V350 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein M351 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein V352 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein R353 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F354 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S355 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein T356 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein Y357 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein F358 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F359 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R360 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N361 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein S362 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein W363 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein Y364 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein W365 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein L366 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Y367 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein E368 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N369 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R370 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N371 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein N372 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R373 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T374 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein R375 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein Y376 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G377 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D378 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P379 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein I380 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q381 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein I382 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L383 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein T384 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G385 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W386 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein P387 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein G388 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I389 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P390 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein T391 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein H392 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N393 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein I394 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D395 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A396 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F397 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V398 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein H399 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I400 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W401 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein T402 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein W403 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein K404 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R405 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein D406 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E407 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R408 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein Y409 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein F410 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F411 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q412 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein G413 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N414 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein Q415 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein Y416 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein W417 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein R418 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein Y419 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein D420 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S421 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D422 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K423 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D424 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q425 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein A426 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L427 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein T428 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E429 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D430 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E431 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q432 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein G433 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K434 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S435 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Y436 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein P437 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein K438 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L439 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein I440 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S441 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein E442 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G443 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F444 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P445 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein G446 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I447 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P448 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein S449 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein P450 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein L451 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D452 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T453 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein A454 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F455 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y456 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein D457 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R458 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein R459 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein Q460 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein K461 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L462 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein I463 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y464 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein F465 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F466 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K467 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E468 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S469 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein L470 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V471 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F472 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A473 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F474 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D475 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V476 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein N477 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R478 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N479 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R480 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein V481 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L482 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein N483 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein S484 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Y485 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein P486 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein K487 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R488 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein I489 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T490 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E491 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V492 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F493 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P494 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein A495 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V496 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein I497 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P498 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein Q499 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein N500 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein H501 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P502 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein F503 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R504 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N505 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein I506 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D507 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S508 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein A509 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y510 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein Y511 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein S512 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Y513 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein A514 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y515 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein N516 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein S517 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein I518 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F519 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F520 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F521 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K522 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G523 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N524 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein A525 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y526 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein W527 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein K528 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V529 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein V530 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein N531 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein D532 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K533 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D534 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K535 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q536 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein Q537 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein N538 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein S539 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein W540 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein L541 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P542 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein A543 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and/or wherein N544 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y of SEQ ID NO:2, in addition to any combination thereof. The present invention also encompasses the use of these MMP-29 hemopexin-like domain amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following MMP-29 hemopexin-like domain conservative amino acid substitutions are encompassed by the present invention: wherein Q346 is substituted with a N; wherein Y347 is either an F, or W; wherein G348 is substituted with either an A, M, S, or T; wherein E349 is substituted with a D; wherein V350 is substituted with either an A, I, or L; wherein M351 is substituted with either an A, G, S, or T; wherein V352 is substituted with either an A, I, or L; wherein R353 is substituted with either a K, or H; wherein F354 is substituted with either a W, or Y; wherein S355 is substituted with either an A, G, M, or T; wherein T356 is substituted with either an A, G, M, or S; wherein Y357 is either an F, or W; wherein F358 is substituted with either a W, or Y; wherein F359 is substituted with either a W, or Y; wherein R360 is substituted with either a K, or H; wherein N361 is substituted with a Q; wherein S362 is substituted with either an A, G, M, or T; wherein W363 is either an F, or Y; wherein Y364 is either an F, or W; wherein W365 is either an F, or Y; wherein L366 is substituted with either an A, I, or V; wherein Y367 is either an F, or W; wherein E368 is substituted with a D; wherein N369 is substituted with a Q; wherein R370 is substituted with either a K, or H; wherein N371 is substituted with a Q; wherein N372 is substituted with a Q; wherein R373 is substituted with either a K, or H; wherein T374 is substituted with either an A, G, M, or S; wherein R375 is substituted with either a K, or H; wherein Y376 is either an F, or W; wherein G377 is substituted with either an A, M, S, or T; wherein D378 is substituted with an E; wherein P379 is a P; wherein I380 is substituted with either an A, V, or L; wherein Q381 is substituted with a N; wherein I382 is substituted with either an A, V, or L; wherein L383 is substituted with either an A, I, or V; wherein T384 is substituted with either an A, G, M, or S; wherein G385 is substituted with either an A, M, S, or T; wherein W386 is either an F, or Y; wherein P387 is a P; wherein G388 is substituted with either an A, M, S, or T; wherein I389 is substituted with either an A, V, or L; wherein P390 is a P; wherein T391 is substituted with either an A, G, M, or S; wherein H392 is substituted with either a K, or R; wherein N393 is substituted with a Q; wherein I394 is substituted with either an A, V, or L; wherein D395 is substituted with an E; wherein A396 is substituted with either a G, I, L, M, S, T, or V; wherein F397 is substituted with either a W, or Y; wherein V398 is substituted with either an A, I, or L; wherein H399 is substituted with either a K, or R; wherein I400 is substituted with either an A, V, or L; wherein W401 is either an F, or Y; wherein T402 is substituted with either an A, G, M, or S; wherein W403 is either an F, or Y; wherein K404 is substituted with either a R, or H; wherein R405 is substituted with either a K, or H; wherein D406 is substituted with an E; wherein E407 is substituted with a D; wherein R408 is substituted with either a K, or H; wherein Y409 is either an F, or W; wherein F410 is substituted with either a W, or Y; wherein F411 is substituted with either a W, or Y; wherein Q412 is substituted with a N; wherein G413 is substituted with either an A, M, S, or T; wherein N414 is substituted with a Q; wherein Q415 is substituted with a N; wherein Y416 is either an F, or W; wherein W417 is either an F, or Y; wherein R418 is substituted with either a K, or H; wherein Y419 is either an F, or W; wherein D420 is substituted with an E; wherein S421 is substituted with either an A, G, M, or T; wherein D422 is substituted with an E; wherein K423 is substituted with either a R, or H; wherein D424 is substituted with an E; wherein Q425 is substituted with a N; wherein A426 is substituted with either a G, I, L, M, S, T, or V; wherein L427 is substituted with either an A, I, or V; wherein T428 is substituted with either an A, G, M, or S; wherein E429 is substituted with a D; wherein D430 is substituted with an E; wherein E431 is substituted with a D; wherein Q432 is substituted with a N; wherein G433 is substituted with either an A, M, S, or T; wherein K434 is substituted with either a R, or H; wherein S435 is substituted with either an A, G, M, or T; wherein Y436 is either an F, or W; wherein P437 is a P; wherein K438 is substituted with either a R, or H; wherein L439 is substituted with either an A, I, or V; wherein I440 is substituted with either an A, V, or L; wherein S441 is substituted with either an A, G, M, or T; wherein E442 is substituted with a D; wherein G443 is substituted with either an A, M, S, or T; wherein F444 is substituted with either a W, or Y; wherein P445 is a P; wherein G446 is substituted with either an A, M, S, or T; wherein I447 is substituted with either an A, V, or L; wherein P448 is a P; wherein S449 is substituted with either an A, G, M, or T; wherein P450 is a P; wherein L451 is substituted with either an A, T, or V; wherein D452 is substituted with an E; wherein T453 is substituted with either an A, G, M, or S; wherein A454 is substituted with either a G, I, L, M, S, T, or V; wherein F455 is substituted with either a W, or Y; wherein Y456 is either an F, or W; wherein D457 is substituted with an E; wherein R458 is substituted with either a K, or H; wherein R459 is substituted with either a K, or H; wherein Q460 is substituted with a N; wherein K461 is substituted with either a R, or H; wherein L462 is substituted with either an A, I, or V; wherein I463 is substituted with either an A, V, or L; wherein Y464 is either an F, or W; wherein F465 is substituted with either a W, or Y; wherein F466 is substituted with either a W, or Y; wherein K467 is substituted with either a R, or H; wherein E468 is substituted with a D; wherein S469 is substituted with either an A, G, M, or T; wherein L470 is substituted with either an A, I, or V; wherein V471 is substituted with either an A, I, or L; wherein F472 is substituted with either a W, or Y; wherein A473 is substituted with either a G, I, L, M, S, T, or V; wherein F474 is substituted with either a W, or Y; wherein D475 is substituted with an E; wherein V476 is substituted with either an A, I, or L; wherein N477 is substituted with a Q; wherein R478 is substituted with either a K, or H; wherein N479 is substituted with a Q; wherein R480 is substituted with either a K, or H; wherein V481 is substituted with either an A, I, or L; wherein L482 is substituted with either an A, I, or V; wherein N483 is substituted with a Q; wherein S484 is substituted with either an A, G, M, or T; wherein Y485 is either an F, or W; wherein P486 is a P; wherein K487 is substituted with either a R, or H; wherein R488 is substituted with either a K, or H; wherein 1489 is substituted with either an A, V, or L; wherein T490 is substituted with either an A, G, M, or S; wherein E491 is substituted with a D; wherein V492 is substituted with either an A, I, or L; wherein F493 is substituted with either a W, or Y; wherein P494 is a P; wherein A495 is substituted with either a G, I, L, M, S, T, or V; wherein V496 is substituted with either an A, I, or L; wherein 1497 is substituted with either an A, V, or L; wherein P498 is a P; wherein Q499 is substituted with a N; wherein N500 is substituted with a Q; wherein H501 is substituted with either a K, or R; wherein P502 is a P; wherein F503 is substituted with either a W, or Y; wherein R504 is substituted with either a K, or H; wherein N505 is substituted with a Q; wherein I506 is substituted with either an A, V, or L; wherein D507 is substituted with an E; wherein S508 is substituted with either an A, G, M, or T; wherein A509 is substituted with either a G, I, L, M, S, T, or V; wherein Y510 is either an F, or W; wherein Y511 is either an F, or W; wherein S512 is substituted with either an A, G, M, or T; wherein Y513 is either an F, or W; wherein A514 is substituted with either a G, I, L, M, S, T, or V; wherein Y515 is either an P, or W; wherein N516 is substituted with a Q; wherein S517 is substituted with either an A, G, M, or T; wherein I518 is substituted with either an A, V, or L; wherein F519 is substituted with either a W, or Y; wherein F520 is substituted with either a W, or Y; wherein F521 is substituted with either a W, or Y; wherein K522 is substituted with either a R, or H; wherein G523 is substituted with either an A, M, S, or T; wherein N524 is substituted with a Q; wherein A525 is substituted with either a G, I, L, M, S, T, or V; wherein Y526 is either an F, or W; wherein W527 is either an F, or Y; wherein K528 is substituted with either a R, or H; wherein V529 is substituted with either an A, I, or L; wherein V530 is substituted with either an A, I, or L; wherein N531 is substituted with a Q; wherein D532 is substituted with an E; wherein K533 is substituted with either a R, or H; wherein D534 is substituted with an E; wherein K535 is substituted with either a R, or H; wherein Q536 is substituted with a N; wherein Q537 is substituted with a N; wherein N538 is substituted with a Q; wherein S539 is substituted with either an A, G, M, or T; wherein W540 is either an F, or Y; wherein L541 is substituted with either an A, I, or V; wherein P542 is a P; wherein A543 is substituted with either a G, I, L, M, S, T, or V; and/or wherein N544 is substituted with a Q of SEQ ID NO:2 in addition to any combination thereof. Other suitable substitutions within the MMP-29 hemopexin-like domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these MMP-29 hemopexin-like domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

For purposes of the present invention, by “at least a portion of” is meant all or any part of the MMP-29 propeptide, catalytic, or hemopexin-like domain defined by the structure coordinates according to Tables IV, V, or VI (e.g., fragments thereof). More preferred are molecules comprising all or any parts of the MMP-29 propeptide, catalytic, or hemopexin-like domain, according to Tables IV, V, or VI, or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said MMP-29 amino acids of not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms.

The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a term that expresses the deviation or variation from a trend or object. For the purposes of the present invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of the AR portion of the complex as defined by the structure coordinates described herein.

A preferred embodiment is a machine-readable data storage medium that is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex that is defined by the structure coordinates of all of the amino acids in Tables IV, V, or VI +/− a root mean square deviation from the backbone atoms of those amino acids of not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms.

The structure coordinates of a MMP-29 propeptide, catalytic, or hemopexin-like domain homology models, including portions thereof, is stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery.

Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Tables IV, V, or VI.

One embodiment utilizes System 10 as disclosed in WO 98/11134, the disclosure of which is incorporated herein by reference in its entirety. Briefly, one version of these embodiments comprises a computer comprising a central processing unit (“CPU”), a working memory which may be, e.g, RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or more keyboards, one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, keyboard may also be used as an input device.

Output hardware, coupled to the computer by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a CRT display terminal for displaying a graphical representation of a region or domain of the present invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.

In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage, and accesses to and from the working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system are included as appropriate throughout the following description of the data storage medium.

For the purpose of the present invention, any magnetic data storage medium which can be encoded with machine-readable data would be sufficient for carrying out the storage requirements of the system. The medium could be a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, containing magnetic domains whose polarity or orientation could be altered magnetically, for example. The medium may also have an opening for receiving the spindle of a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as the system described herein.

Another example of a suitable storage medium which could also be encoded with such machine-readable data, or set of instructions, which could be carried out by a system such as the system described herein, could be an optically-readable data storage medium. The medium could be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable. The medium preferably has a suitable substrate, which may be conventional, and a suitable coating , which may be conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of the coating. A protective coating, which preferably is substantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating has no pits, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser. The orientation of the domains can be read by measuring the polarization of laser light reflected from the coating. The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable of displaying the three dimensional structure of the MMP-29 propeptide, catalytic, or hemopexin-like domain homology models, or portions thereof and their structurally similar homologues is stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure. Such data may be used for a variety of purposes, such as drug discovery.

For the first time, the present invention permits the use of structure-based or rational drug design techniques to design, select, and synthesize chemical entities that are capable of modulating the biological function of MMP-29.

Accordingly, the present invention is also directed to the design of small molecules which imitates the structure of the MMP-29 propeptide, catalytic, and/or hemopexin-like domain (SEQ ID NO:75, 76, and/or 77), or a portion thereof, in accordance with the structure coordinates provided in Tables IV, V, and/or VI. Alternatively, the present invention is directed to the design of small molecules which may bind to at least part of the MMP-29 propeptide, catalytic, and/or hemopexin-like domain (SEQ ID NO:75, 76, and/or 77), or some portion thereof. For purposes of this invention, by MMP-29 propeptide, catalytic, and/or hemopexin-like domain, it is also meant to include mutants or homologues thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to SEQ ID NO:75, 76, and/or 77. In this context, the term “small molecule” may be construed to mean any molecule described known in the art or described elsewhere herein, though may include, for example, peptides, chemicals, carbohydrates, nucleic acids, PNAs, and any derivatives thereof.

The three-dimensional model structure of the MMP-29 will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.

For example, test compounds can be modeled that fit spatially into the catalytic domain in MMP-29 embodied by the sequence at amino C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2, in accordance with the structural coordinates of Table V.

Structure coordinates of the catalytic domain in MMP-29 defined by the amino acids at amino acid C117, at amino acid H283, at amino acid H287, at amino acid H293, at amino acid Q247, at amino acid E248, at amino acid F249, at amino acid A250, at amino acid I258, at amino acid Y273, at amino acid G274, and/or at amino acid Q302 of SEQ ID NO:2, can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential MMP-29 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction with, the three-dimensional structural model can be employed to design or select compounds as potential MMP-29 modulators. Compounds identified as potential MMP-29 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the MMP-29, or in characterizing the ability of MMP-29 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential MMP-29 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids at amino acid positions, C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and/or Q302 of SEQ ID NO:2, or amino acids G161 through E330 in accordance with the structure coordinates of Table V.

Structure coordinates of the propeptide domain in MMP-29 defined by the amino acids at amino acid A48 through P120 of SEQ ID NO:2, can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential MMP-29 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction with, the three-dimensional structural model can be employed to design or select compounds as potential MMP-29 modulators. Compounds identified as potential MMP-29 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the MMP-29, or in characterizing the ability of MMP-29 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential MMP-29 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids at amino acid positions, A48 through P120 of SEQ ID NO:2 in accordance with the structure coordinates of Table IV.

Structure coordinates of the hemopexin-like domain in MMP-29 defined by the amino acids at amino acid Q346 through N544 of SEQ ID NO:2, can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential MMP-29 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction with, the three-dimensional structural model can be employed to design or select compounds as potential MMP-29 modulators. Compounds identified as potential MMP-29 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the MMP-29, or in characterizing the ability of MMP-29 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential MMP-29 modulators include, but are hot limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids at amino acid positions, Q346 through N544 of SEQ ID NO:2 in accordance with the structure coordinates of Table VI.

For example, test compounds can be modeled that fit spatially into the S1 pocket in MMP-29 embodied by the sequence at amino Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2, in accordance with the structural coordinates of Table V.

Structure coordinates of the S1 pocket in MMP-29 defined by the amino acids at amino acid Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2, can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential MMP-29 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction with, the three-dimensional structural model can be employed to design or select compounds as potential MMP-29 modulators. Compounds identified as potential MMP-29 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the MMP-29, or in characterizing the ability of MMP-29 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential MMP-29 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids at amino acid positions, Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2 in accordance with the structure coordinates of Table V.

However, as will be understood by those of skill in the art upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.

For example, a number of computer modeling systems are available in which the sequence of the MMP-29 and the MMP-29 structure (i.e., atomic coordinates of MMP-29 and/or the atomic coordinates of the active site domain as provided in Table IV) can be input. This computer system then generates the structural details of one or more these regions in which a potential MMP-29 modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with MMP-29. In addition, the compound must be able to assume a conformation that allows it to associate with MMP-29. Some modeling systems estimate the potential inhibitory or binding effect of a potential MMP-29 modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their ability to associate with a given protein target are also well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in the active site domain of MMP-29. Docking is accomplished using software such as INSIGHTII, QUANTA and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as MMFF, CHARMM and AMBER. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et al. 1982).

Upon selection of preferred chemical entities or fragments, their relationship to each other and MMP-29 can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to CAVEAT (Bartlett et al. 1989) and 3D Database systems (Martin 1992).

Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).

In addition, MMP-29 is overall well suited to modern methods including combinatorial chemistry.

Programs such as DOCK (Kuntz et. al. 1982) can be used with the atomic coordinates from the MMP-29 homology models to identify potential ligands from databases or virtual databases which potentially bind the in the active site region, and which may therefore be suitable candidates for synthesis and testing. The computer programs may utilize a combination of the following steps: (a) selection of fragments or chemical entities from a database and then positioning the chemical entity in one or more orientations within the MMP29 active site defined by residues C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and Q302; (b) characterization of the structural and chemical features of the chemical entity and active site including van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interactions; (c) search databases for molecular fragments which can be joined to or replace the docked chemical entity and spatially fit into regions defined by the said MMP29 active site; and/or (d) evaluate the docked chemical entity and fragments using a combination of scoring schemes which account for van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic interactions

Examples of databases that may be used include ACD (Molecular Designs Limited), Aldrich (Aldrich Chemical Company), NCI (National Cancer Institute), Maybridge(Maybridge Chemical Company Ltd), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical Abstract Service), Derwent (Derwent Information Limited), in addition to others known in the art.

Additionally, the three-dimensional homology model of MMP-29 will aid in the design of mutants with altered biological activity.

The following are encompassed by the present invention: a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain models according to Tables IV, V, and/or VI or a homologue of said model, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of the complex of not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms; and a machine-readable data storage medium, wherein said molecule is defined by the set of structure coordinates of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain models according to Tables IV, V, and/or VI, or a homologue of said molecule, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms; a model comprising all or any part of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain models defined by structure coordinates according to Tables IV, V, and/or VI, or a mutant or homologue of said molecule or molecular complex.

In a further embodiment, the following are encompassed by the present invention: a method for identifying a mutant of MMP-29 with altered biological properties, function, or reactivity, the method comprising any combination of steps of: use of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain models or a homologue of said models according to Tables IV, V, and/or VI, for the design of protein mutants with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; and use of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain models or a homologue of said models, for the design of a protein with mutations in the propeptide domain comprised of the amino acids at amino acid A48 thru P120 according to Table IV with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; or for the design of a protein with mutations in the catalytic domain comprised of the amino acids at amino acid G161 through E330 according to Table V with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; or for the design of a protein with mutations in the catalytic domain comprised of the amino acids at amino acid Q346 through N544 according to Table VI with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein

In further preferred embodiments, the following are encompassed by the present invention: a method for identifying modulators of MMP-29 biological properties, function, or reactivity, the method comprising any combination of steps of: modeling test compounds that overlay spatially into the catalytic domain defined by all or any portion of residues at amino acid G161 through E330, or preferably as defined by residues C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and Q302 and of the three-dimensional structural model according to Table V, or using a homologue or portion thereof.

In further preferred embodiments, the following are encompassed by the present invention: a method for identifying modulators of MMP-29 biological properties, function, or reactivity, the method comprising any combination of steps of: modeling test compounds that overlay spatially into the SI pocket defined by all or any portion of residues at amino acid Q247, E248, F249, A250, 1258, Y273, G274, and Q302 and of the three-dimensional structural model according to Table V, or using a homologue or portion thereof.

The present invention encompasses using the structure coordinates as set forth herein to identify structural and chemical features of the MMP-29 polypeptide; employing identified structural or chemical features to design or select compounds as potential MMP-29 modulators; employing the three-dimensional structural models described herien to design or select compounds as potential MMP-29 modulators; synthesizing the potential MMP-29 modulators; screening the potential MMP-29 modulators in an assay characterized by binding of a protein to the MMP-29; selecting the potential MMP-29 modulator from a database; designing the MMP-29 modulator de novo; and/or designing said MMP-29 modulator from a known modulator activity.

Thus, one embodiment of the invention relaties to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the MMP-29 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. Accordingly, the cDNA sequence for MMP-29 depicted in FIG. 1A-B as well as vectors and host cells expressing the MMP-29 protein or peptides thereof are useful in methods of identifying agents which alter or inhibit MMP-29 activities through modulation of the catalytic region(s). High-throughput screening assays such as proximity-based assays can also be developed using radiolabeled or fluorescent-labeled molecules.

The invention also relates to in silico screening methods including in silico docking and methods of structure based drug design which utilize the three dimensional structural coordinates of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain (Tables IV, V, and/or VI). Also provided are methods of identifying modulators of MMP-29 that include modulator building or searching utilizing computer programs and algorithms. In an embodiment of the invention a method is provided for designing potential modulators of MMP-29 comprising any combination of steps which utilize said three dimensional structure to design or select potential modulators.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:1 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1693 of SEQ ID NO:1, b is an integer between 15 to 1707, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:1, and where b is greater than or equal to a+14.

In one embodiment, a MMP-29 polypeptide comprises a portion of the amino sequence depicted in FIG. 1A–B. In another embodiment, a MMP-29 polypeptide comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the amino sequence depicted in FIG. 1A–B. In further embodiments, the following MMP-29 polypeptide fragments are specifically excluded from the present invention: DTAFYDRRQKLIYFFKESLV (SEQ ID NO:78); AFDVNRNRVLNSYPKRITEVFPAVIPQNHPFRNIDSAYYSYAYNSIFFKGNAY WKVVNDKDKQQNSWLPANGLFPKKFISEKWFDVCDVHISTLNM (SEQ ID NO:79); or LLKVAVHEIGH (SEQ ID NO:80).

TABLE I ATCC NT Total 5' NT Total Deposit SEQ NT of Start 3' NT AA AA Gene CDNA No. Z and ID. Seq of Codon of Seq ID of No. CloneID Date Vector No. X Clone of ORF ORF No. Y ORF 1. MMP-29 XXXXX PSport 1 1 1707 1 1707 2 569 (BSG-26) Xx/Xx/Xx

Table I summarizes the information corresponding to each “Gene No.” described above. The nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the “cDNA clone ID” identified in Table I and, in some cases, from additional related DNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually several overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:1.

The cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in “ATCC Deposit No:Z and Date.” “Vector” refers to the type of vector contained in the cDNA Clone ID.

“Total NT Seq. Of Clone” refers to the total number of nucleotides in the clone contig identified by “Gene No.” The deposited clone may contain all or most of the sequence of SEQ ID NO:1. The nucleotide position of SEQ ID NO:1 of the putative start codon (methionine) is identified as “5′ NT of Start Codon of ORF.”

The translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:2,” although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

The total number of amino acids within the open reading frame of SEQ ID NO:2 is identified as “Total AA of ORF”.

SEQ ID NO:1 (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:2 (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further herein. For instance, SEQ ID NO:1 is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:1 or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:2 may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the proteins encoded by the cDNA clones identified in Table I.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides may cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:1 and the predicted translated amino acid sequence identified as SEQ ID NO:2, but also a sample of plasmid DNA containing a cDNA of the invention deposited with the ATCC, as set forth in Table I. The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited cDNA, collecting the protein, and determining its sequence.

The present invention also relates to the genes corresponding to SEQ ID NO:1, or the deposited clone. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Also provided in the present invention are species homologs, allelic variants, and/or orthologs. The skilled artisan could, using procedures well-known in the art, obtain the polynucleotide sequence corresponding to full-length genes (including, but not limited to the full-length coding region), allelic variants, splice variants, orthologs, and/or species homologues of genes corresponding to SEQ ID NO:1, or a deposited clone, relying on the sequence from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologues may be isolated and identified by making suitable probes or primers which correspond to the 5′, 3′, or internal regions of the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

The polypeptides may be in the form of the protein, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31–40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using protocols described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the full-length form of the protein.

The present invention provides a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:1, and/or a cDNA provided in ATCC Deposit No. Z:. The present invention also provides a polypeptide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:2, and/or a polypeptide encoded by the cDNA provided in ATCC Deposit NO:Z. The present invention also provides polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:2, and/or a polypeptide sequence encoded by the cDNA contained in ATCC Deposit No:Z.

Preferably, the present invention is directed to a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:1, and/or a cDNA provided in ATCC Deposit No.: that is less than, or equal to, a polynucleotide sequence that is 5 mega basepairs, 1 mega basepairs, 0.5 mega basepairs, 0.1 mega basepairs, 50,000 basepairs, 20,000 basepairs, or 10,000 basepairs in length.

The present invention encompasses polynucleotides with sequences complementary to those of the polynucleotides of the present invention disclosed herein. Such sequences may be complementary to the sequence disclosed as SEQ ID NO:1, the sequence contained in a deposit, and/or the nucleic acid sequence encoding the sequence disclosed as SEQ ID NO:2.

The present invention also encompasses polynucleotides capable of hybridizing, preferably under reduced stringency conditions, more preferably under stringent conditions, and most preferably under highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in Table II below: highly stringent conditions are those that are at least as stringent as, for example, conditions A–F; stringent conditions are at least as stringent as, for example, conditions G–L; and reduced stringency conditions are at least as stringent as, for example, conditions M–R.

TABLE II Poly- Hybrid Hybridization Wash Stringency nucleotide Length Temperature Temperature Condition Hybrid± (bp) ‡ and Buffer † and Buffer † A DNA:DNA > or equal to 65° C.; 1xSSC- 65° C.; 50 or- 42° C.; 0.3xSSC 1xSSC, 50% formamide B DNA:DNA < 50 Tb*; 1xSSC Tb*; 1xSSC C DNA:RNA > or equal to 67° C.; 1xSSC- 67° C.; 50 or- 45° C.; 0.3xSSC 1xSSC, 50% formamide D DNA:RNA < 50 Td*; 1xSSC Td*; 1xSSC E RNA:RNA > or equal to 70° C.; 1xSSC- 70° C.; 50 or- 50° C.; 0.3xSSC 1xSSC, 50% formamide F RNA:RNA < 50 Tf*; 1xSSC Tf*; 1xSSC G DNA:DNA > or equal to 65° C.; 4xSSC- 65° C.; 1xSSC 50 or- 45° C.; 4xSSC, 50% formamide H DNA:DNA < 50 Th*; 4xSSC Th*; 4xSSC I DNA:RNA > or equal to 67° C.; 4xSSC- 67° C.; 1xSSC 50 or- 45° C.; 4xSSC, 50% formamide J DNA:RNA < 50 Tj*; 4xSSC Tj*; 4xSSC K RNA:RNA > or equal to 70° C.; 4xSSC- 67° C.; 1xSSC 50 or- 40° C.; 6xSSC, 50% formamide L RNA:RNA < 50 Tl*; 2xSSC Tl*; 2xSSC M DNA:DNA > or equal to 50° C.; 4xSSC- 50° C.; 2xSSC 50 or- 40° C. 6xSSC, 50% formamide N DNA:DNA < 50 Tn*; 6xSSC Tn*; 6xSSC O DNA:RNA > or equal to 55° C.; 4xSSC- 55° C.; 2xSSC 50 or- 42° C.; 6xSSC, 50% formamide P DNA:RNA < 50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA > or equal to 60° C.; 4xSSC- 60° C.; 2xSSC 50 or- 45° C.; 6xSSC, 50% formamide R RNA:RNA < 50 Tr*; 4xSSC Tr*; 4xSSC

‡—The “hybrid length” is the anticipated length for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucleotide of unknown sequence, the hybrid is assumed to be that of the hybridizing polynucleotide of the present invention. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. Methods of aligning two or more polynucleotide sequences and/or determining the percent identity between two polynucleotide sequences are well known in the art (e.g., MegAlign program of the DNA*Star suite of programs, etc).

†—SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. The hydridizations and washes may additionally include 5× Denhardt's reagent, 0.5–1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide.

*Tb—Tr: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5–10° C. less than the melting temperature Tm of the hybrids there Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.)=81.5+16.6(log₁₀[Na+])+0.41(% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([NA+] for 1×SSC=0.165 M).

±—The present invention encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a PNA, or a modified polynucleotide. Such modified polynucleotides are known in the art and are more particularly described elsewhere herein.

Additional examples of stringency conditions for polynucleotide hybridization are provided, for example, in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel et al., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3–6.4, which are hereby incorporated by reference herein.

Preferably, such hybridizing polynucleotides have at least 70% sequence identity (more preferably, at least 80% identity; and most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which they hybridize, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The determination of identity is well known in the art, and discussed more specifically elsewhere herein.

The invention encompasses the application of PCR methodology to the polynucleotide sequences of the present invention, the clone deposited with the ATCC, and/or the cDNA encoding the polypeptides of the present invention. PCR techniques for the amplification of nucleic acids are described in U.S. Pat. No. 4,683,195 and Saiki et al., Science, 239:487–491 (1988). PCR, for example, may include the following steps, of denaturation of template nucleic acid (if double-stranded), annealing of primer to target, and polymerization. The nucleic acid probed or used as a template in the amplification reaction may be genomic DNA, cDNA, RNA, or a PNA. PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA. References for the general use of PCR techniques, including specific method parameters, include Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, Stockton Press, NY, 1989; Ehrlich et al., Science, 252:1643–1650, (1991); and “PCR Protocols, A Guide to Methods and Applications”, Eds., Innis et al., Academic Press, New York, (1990).

Signal Sequences

The present invention also encompasses mature forms of the polypeptide comprising, or alternatively consisting of, the polypeptide sequence of SEQ ID NO:2, the polypeptide encoded by the polynucleotide described as SEQ ID NO:1, and/or the polypeptide sequence encoded by a cDNA in the deposited clone. The present invention also encompasses polynucleotides encoding mature forms of the present invention, such as, for example the polynucleotide sequence of SEQ ID NO:1, and/or the polynucleotide sequence provided in a cDNA of the deposited clone.

According to the signal hypothesis, proteins secreted by eukaryotic cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most eukaryotic cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.

Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271–286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683–4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues −13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75–80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.

The established method for identifying the location of signal sequences, in addition, to their cleavage sites has been the SignalP program (v1.1) developed by Henrik Nielsen et al., Protein Engineering 10:1–6 (1997). The program relies upon the algorithm developed by von Heinje, though provides additional parameters to increase the prediction accuracy.

More recently, a hidden Markov model has been developed (H. Neilson, et al., Ismb 1998;6:122–30), which has been incorporated into the more recent SignalP (v2.0). This new method increases the ability to identify the cleavage site by discriminating between signal peptides and uncleaved signal anchors. The present invention encompasses the application of the method disclosed therein to the prediction of the signal peptide location, including the cleavage site, to any of the polypeptide sequences of the present invention.

As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the polypeptide of the present invention may contain a signal sequence. Polypeptides of the invention which comprise a signal sequence have an N-terminus beginning within 5 residues (i.e., + or −5 residues, or preferably at the −5, −4, −3, −2, −1, +1, +2, +3, +4, or +5 residue) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:1 and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as described below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention also encompasses variants (e.g., allelic variants, orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQ ID NO:1, the complementary strand thereto, and/or the cDNA sequence contained in the deposited clone.

The present invention also encompasses variants of the polypeptide sequence, and/or fragments therein, disclosed in SEQ ID NO:2, a polypeptide encoded by the polynucleotide sequence in SEQ ID NO:1, and/or a polypeptide encoded by a cDNA in the deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.

Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a MMP-29 related polypeptide having an amino acid sequence as shown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (b) a nucleotide sequence encoding a mature MMP-29 related polypeptide having the amino acid sequence as shown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (c) a nucleotide sequence encoding a biologically active fragment of a MMP-29 related polypeptide having an amino acid sequence shown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (d) a nucleotide sequence encoding an antigenic fragment of a MMP-29 related polypeptide having an amino acid sequence sown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (e) a nucleotide sequence encoding a MMP-29 related polypeptide comprising the complete amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (f) a nucleotide sequence encoding a mature MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (g) a nucleotide sequence encoding a biologically active fragment of a MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (h) a nucleotide sequence encoding an antigenic fragment of a MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (I) a nucleotide sequence complimentary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.

The present invention is also directed to polynucleotide sequences which comprise, or alternatively consist of, a polynucleotide sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1% 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.

Another aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively, consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a MMP-29 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (b) a nucleotide sequence encoding a mature MMP-29 related polypeptide having the amino acid sequence as shown in the sequence listing and descried in Table I; (c) a nucleotide sequence encoding a biologically active fragment of a MMP-29 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (d) a nucleotide sequence encoding an antigenic fragment of a MMP-29 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (e) a nucleotide sequence encoding a MMP-29 related polypeptide comprising the complete amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I; (f) a nucleotide sequence encoding a mature MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I: (g) a nucleotide sequence encoding a biologically active fragment of a MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I; (h) a nucleotide sequence encoding an antigenic fragment of a MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC deposit and described in Table I; (i) a nucleotide sequence complimentary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h) above.

The present invention is also directed to nucleic acid molecules which comprise, or alternatively, consist of, a nucleotide sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1% 99.2% 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.

The present invention encompasses polypeptide sequences which comprise, or alternatively consist of, an amino acid sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3% 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, the following non-limited examples, the polypeptide sequence identified as SEQ ID NO:2, the polypeptide sequence encoded by a cDNA provided in the deposited clone, and/or polypeptide fragments of any of the polypeptides provided herein. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.

The present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3% 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, the polypeptide sequence shown in SEQ ID NO:2, a polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:1, a polypeptide sequence encoded by the cDNA in cDNA plasmid:Z, and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these polypeptides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompasses by the present invention, as are the polypeptides encoded by these polynucleotides.

By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referenced in Table I, the ORF (open reading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673–4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189–191, (1992). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. However, the CLUSTALW algorithm automatically converts U's to T's when comparing RNA sequences to DNA sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).

The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polynucleotide alignment. Percent identity calculations based upon global polynucleotide alignments are often preferred since they reflect the percent identity between the polynucleotide molecules as a whole (i.e., including any polynucleotide overhangs, not just overlapping regions), as opposed to, only local matching polynucleotides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This corrected score may be used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALW alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the CLUSTALW alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2% 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for instance, an amino acid sequence referenced in Table 1 (SEQ ID NO:2) or to the amino acid sequence encoded by cDNA contained in a deposited clone, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673–4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189–191, (1992). In a sequence alignment the query and subject sequences are both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=BLOSUM, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).

The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of N- or C-terminal deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polypeptide alignment. Percent identity calculations based upon global polypeptide alignments are often preferred since they reflect the percent identity between the polypeptide molecules as a whole (i.e., including any polypeptide overhangs, not just overlapping regions), as opposed to, only local matching polypeptides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for N- and C-terminal truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what may be used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the CLUSTALW alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence, which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the CLUSTALW alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.

In addition to the above method of aligning two or more polynucleotide or polypeptide sequences to arrive at a percent identity value for the aligned sequences, it may be desirable in some circumstances to use a modified version of the CLUSTALW algorithm which takes into account known structural features of the sequences to be aligned, such as for example, the SWISS-PROT designations for each sequence. The result of such a modifed CLUSTALW algorithm may provide a more accurate value of the percent identity for two polynucleotide or polypeptide sequences. Support for such a modified version of CLUSTALW is provided within the CLUSTALW algorithm and would be readily appreciated to one of skill in the art of bioinformatics.

The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5–10, 1–5, or 1–2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the mRNA to those preferred by a bacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984–2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8–10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199–216 (1988)).

Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem. . . . 268:22105–22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the protein will likely be retained when less than the majority of the residues of the protein are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.

Alternatively, such N-terminus or C-terminus deletions of a polypeptide of the present invention may, in fact, result in a significant increase in one or more of the biological activities of the polypeptide(s). For example, biological activity of many polypeptides are governed by the presence of regulatory domains at either one or both termini. Such regulatory domains effectively inhibit the biological activity of such polypeptides in lieu of an activation event (e.g., binding to a cognate ligand or receptor, phosphorylation, proteolytic processing, etc.). Thus, by eliminating the regulatory domain of a polypeptide, the polypeptide may effectively be rendered biologically active in the absence of an activation event.

Thus, the invention further includes polypeptide variants that show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247:1306–1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081–1085 (1989).) The resulting mutant molecules can then be tested for biological activity.

As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.

The invention encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the polypeptide of the present invention. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics (e.g., chemical properties). According to Cunningham et al above, such conservative substitutions are likely to be phenotypically silent. Additional guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306–1310(1990).

Tolerated conservative amino acid substitutions of the present invention involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservative substitutions provided in Table IV below.

TABLE IV For Amino Acid Code Replace with any of: Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D- Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro, L-1-thioazolidine-4-carboxylic acid, D- or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Aside from the uses described above, such amino acid substitutions may also increase protein or peptide stability. The invention encompasses amino acid substitutions that contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the protein or peptide sequence. Also included are substitutions that include amino acid residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.

Both identity and similarity can be readily calculated by reference to the following publications: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Informatics Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

In addition, the present invention also encompasses substitution of amino acids based upon the probability of an amino acid substitution resulting in conservation of function. Such probabilities are determined by aligning multiple genes with related function and assessing the relative penalty of each substitution to proper gene function. Such probabilities are often described in a matrix and are used by some algorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percent similarity wherein similarity refers to the degree by which one amino acid may substitute for another amino acid without lose of function. An example of such a matrix is the PAM250 or BLOSUM62 matrix.

Aside from the canonical chemically conservative substitutions referenced above, the invention also encompasses substitutions which are typically not classified as conservative, but that may be chemically conservative under certain circumstances. Analysis of enzymatic catalysis for proteases, for example, has shown that certain amino acids within the active site of some enzymes may have highly perturbed pKa's due to the unique microenvironment of the active site. Such perturbed pKa's could enable some amino acids to substitute for other amino acids while conserving enzymatic structure and function. Examples of amino acids that are known to have amino acids with perturbed pKa's are the Glu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, the His-159 residue of Papain, etc. The conservation of function relates to either anomalous protonation or anomalous deprotonation of such amino acids, relative to their canonical, non-perturbed pKa. The pKa perturbation may enable these amino acids to actively participate in general acid-base catalysis due to the unique ionization environment within the enzyme active site. Thus, substituting an amino acid capable of serving as either a general acid or general base within the microenvironment of an enzyme active site or cavity, as may be the case, in the same or similar capacity as the wild-type amino acid, would effectively serve as a conservative amino substitution.

Besides conservative amino acid substitution, variants of the present invention include, but are not limited to, the following: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331–340 (1967); Robbins et al., Diabetes 36: 838–845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307–377 (1993).)

Moreover, the invention further includes polypeptide variants created through the application of molecular evolution (“DNA Shuffling”) methodology to the polynucleotide disclosed as SEQ ID NO:1, the sequence of the clone submitted in a deposit, and/or the cDNA encoding the polypeptide disclosed as SEQ ID NO:2. Such DNA Shuffling technology is known in the art and more particularly described elsewhere herein (e.g., WPC, Stemmer, PNAS, 91:10747, (1994)), and in the Examples provided herein).

A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1–5, 5–10, 5–25, 5–50, 10–50 or 50–150, conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is directed to polynucleotide fragments of the polynucleotides of the invention, in addition to polypeptides encoded therein by said polynucleotides and/or fragments.

In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:1 or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2. The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID NO:1. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1–50, 51–100, 101–150, 151–200, 201–250, 251–300, 301–350, 351–400, 401–450, 451–500, 501–550, 551–600, 651–700, 701–750, 751–800, 800–850, 851–900, 901–950, 951–1000, 1001–1050, 1051–1100, 1101–1150, 1151–1200, 1201–1250, 1251–1300, 1301–1350, 1351–1400, 1401–1450, 1451–1500, 1501–1550, 1551–1600, 1601–1650, 1651–1700, 1701–1750, 1751–1800, 1801–1850, 1851–1900, 1901–1950, 1951–2000, or 2001 to the end of SEQ ID NO:1, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Also encompassed by the present invention are polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions, as are the polypeptides encoded by these polynucleotides.

In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:2 or encoded by the cDNA contained in a deposited clone. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1–20, 21–40, 41–60, 61–80, 81–100, 102–120, 121–140, 141–160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Preferred polypeptide fragments include the full-length protein. Further preferred polypeptide fragments include the full-length protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1–60, can be deleted from the amino terminus of the full-length polypeptide. Similarly, any number of amino acids, ranging from 1–30, can be deleted from the carboxy terminus of the full-length protein. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO:2 falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.

In a preferred embodiment, the functional activity displayed by a polypeptide encoded by a polynucleotide fragment of the invention may be one or more biological activities typically associated with the full-length polypeptide of the invention. Illustrative of these biological activities includes the fragments ability to bind to at least one of the same antibodies which bind to the full-length protein, the fragments ability to interact with at lease one of the same proteins which bind to the full-length, the fragments ability to elicit at least one of the same immune responses as the full-length protein (i.e., to cause the immune system to create antibodies specific to the same epitope, etc.), the fragments ability to bind to at least one of the same polynucleotides as the full-length protein, the fragments ability to bind to a receptor of the full-length protein, the fragments ability to bind to a ligand of the full-length protein, and the fragments ability to multimerize with the full-length protein. However, the skilled artisan would appreciate that some fragments may have biological activities which are desirable and directly inapposite to the biological activity of the full-length protein. The functional activity of polypeptides of the invention, including fragments, variants, derivatives, and analogs thereof can be determined by numerous methods available to the skilled artisan, some of which are described elsewhere herein.

The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:1 or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:1), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998–4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131–5135 (1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length, or longer. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767–778 (1984); Sutcliffe et al., Science 219:660–666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910–914; and Bittle et al., J. Gen. Virol. 66:2347–2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347–2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 pg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84–86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fe fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958–3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972–897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724–33 (1997); Harayama, Trends Biotechnol. 16(2):76–82 (1998); Hansson, et al., J. Mol. Biol. 287:265–76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308–13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:1 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:Y, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Moreover, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules, as well as, antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation of the animal or plant, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. . . . 24:316–325 (1983)). Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.

Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60–69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547–1553 (1992).

Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homologue of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologues of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6M, 5×10-7 M, 107 M, 5×10-8 M, 10-8 M, 5×10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, or 10-15 M.

The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981–1988 (1998); Chen et al., Cancer Res. 58(16):3668–3678 (1998); Harrop et al., J. Immunol. 161(4):1786–1794 (1998); Zhu et al., Cancer Res. 58(15):3209–3214 (1998); Yoon et al., J. Immunol. 160(7):3170–3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237–247 (1998); Pitard et al., J. Immunol. Methods 205(2):177–190 (1997); Liautard et al., Cytokine 9(4):233–241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295–11301 (1997); Taryman et al., Neuron 14(4):755–762 (1995); Muller et al., Structure 6(9):1153–1167 (1998); Bartunek et al., Cytokine 8(1):14–20 (1996) (which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).

As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

The antibodies of the present invention may be generated by any suitable method known in the art.

The antibodies of the present invention may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed. (1988); and Current Protocols, Chapter 2; which are hereby incorporated herein by reference in its entirety). In a preferred method, a preparation of the MMP-29 protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. The administration of the polypeptides of the present invention may entail one or more injections of an immunizing agent and, if desired, an adjuvant. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art. For the purposes of the invention, “immunizing agent” may be defined as a polypeptide of the invention, including fragments, variants, and/or derivatives thereof, in addition to fusions with heterologous polypeptides and other forms of the polypeptides described herein.

Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections, though they may also be given intramuscularly, and/or through IV). The immunizing agent may include polypeptides of the present invention or a fusion protein or variants thereof. Depending upon the nature of the polypeptides (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point etc.), it may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Such conjugation includes either chemical conjugation by derivitizing active chemical functional groups to both the polypeptide of the present invention and the immunogenic protein such that a covalent bond is formed, or through fusion-protein based methodology, or other methods known to the skilled artisan. Examples of such immunogenic proteins include, but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Additional examples of adjuvants which may be employed includes the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

The antibodies of the present invention may comprise monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed. (1988), by Hammerling, et al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y., pp. 563–681 (1981); Köhler et al., Eur. J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976), or other methods known to the artisan. Other examples of methods which may be employed for producing monoclonal antibodies includes, but are not limited to, the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026–2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77–96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

In a hybridoma method, a mouse, a humanized mouse, a mouse with a human immune system, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include polypeptides of the present invention or a fusion protein thereof. Preferably, the immunizing agent consists of an MMP-29 polypeptide or, more preferably, with a MMP-29 polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59–103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. More preferred are the parent myeloma cell line (SP2O) as provided by the ATCC. As inferred throughout the specification, human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51–63).

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptides of the present invention. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay (ELISA). Such techniques are known in the art and within the skill of the artisan. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollart, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra, and/or according to Wands et al. (Gastroenterology 80:225–232 (1981)). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The skilled artisan would acknowledge that a variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hydridomas. For example, the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. In this context, the term “monoclonal antibody” refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone. The DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources). The hydridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transformed into host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al, supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563–681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples described herein. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41–50 (1995); Ames et al., J. Immunol. Methods 184:177–186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952–958 (1994); Persic et al., Gene 187 9–18 (1997); Burton et al., Advances in Immunology 57:191–280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864–869 (1992); and Sawai et al., AJRI 34:26–34 (1995); and Better et al., Science 240:1041–1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46–88 (1991); Shu et al., PNAS 90:7995–7999 (1993); and Skerra et al., Science 240:1038–1040 (1988).

For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191–202; Cabilly et al., Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489–498 (1991); Studnicka et al., Protein Engineering 7(6):805–814 (1994); Roguska. et al., PNAS 91:969–973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the methods of Winter and co-workers (Jones et al., Nature, 321:522–525 (1986); Reichmann et al., Nature, 332:323–327 (1988); Verhoeyen et al., Science, 239:1534–1536 (1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possible some FR residues are substituted from analogous sites in rodent antibodies.

In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522–525 (1986); Riechmann et al., Nature 332:323–329 (1988)1 and Presta, Curr. Op. Struct. Biol., 2:593–596 (1992).

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. The techniques of cole et al., and Boerder et al., are also available for the preparation of human monoclonal antibodies (cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol., 147(1):86–95, (1991)).

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65–93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Pat. No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and creation of an antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in the following scientific publications: Marks et al., Biotechnol., 10:779–783 (1992); Lonberg et al., Nature 368:856–859 (1994); Fishwild et al., Nature Biotechnol., 14:845–51 (1996); Neuberger, Nature Biotechnol., 14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65–93 (1995).

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899–903 (1988)).

Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437–444; (1989) and Nissinoff, J. Immunol. 147(8):2429–2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.

Such anti-idiotypic antibodies capable of binding to the MMP-29 polypeptide can be produced in a two-step procedure. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones that produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies.

The antibodies of the present invention may be bispecific antibodies. Bispecific antibodies are monoclonal, Preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present invention, one of the binding specificities may be directed towards a polypeptide of the present invention, the other may be for any other antigen, and preferably for a cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537–539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655–3659 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh et al., Meth. In Enzym., 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360; WO 92/20373; and EP03089). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioester bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:Y.

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457–479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851–855 (1984); Neuberger et al., Nature 312:604–608 (1984); Takeda et al., Nature 314:452–454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423–42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879–5883 (1988); and Ward et al., Nature 334:544–54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038–1041 (1988)).

More preferably, a clone encoding an antibody of the present invention may be obtained according to the method described in the Example section herein.

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101–3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503–5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355–359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51–544 (1987)).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1–2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488–505; Wu and Wu, Biotherapy 3:87–95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573–596 (1993); Mulligan, Science 260:926–932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191–217 (1993); May 1993, TIB TECH 11(5):155–215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91–99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428–1432 (1992); Fell et al., J. Immunol. 146:2446–2452(1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535–10539 (1991); Zheng et al., J. Immunol. 154:5590–5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337–11341(1992) (said references incorporated by reference in their entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84–86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958–3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting the Fe part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fe portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52–58 (1995); Johanson et al., J. Biol. Chem. 270:9459–9471 (1995).

Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821–824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567–1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243–56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623–53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475–506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303–16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119–58 (1982).

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

The present invention also encompasses the creation of synthetic antibodies directed against the polypeptides of the present invention. One example of synthetic antibodies is described in Radrizzani, M., et al., Medicina, (Aires), 59(6):753–8, (1999)). Recently, a new class of synthetic antibodies has been described and are referred to as molecularly imprinted polymers (MIPs) (Semorex, Inc.). Antibodies, peptides, and enzymes are often used as molecular recognition elements in chemical and biological sensors. However, their lack of stability and signal transduction mechanisms limits their use as sensing devices. Molecularly imprinted polymers (MIPs) are capable of mimicking the function of biological receptors but with less stability constraints. Such polymers provide high sensitivity and selectivity while maintaining excellent thermal and mechanical stability. MIPs have the ability to bind to small molecules and to target molecules such as organics and proteins' with equal or greater potency than that of natural antibodies. These “super” MIPs have higher affinities for their target and thus require lower concentrations for efficacious binding.

During synthesis, the MIPs are imprinted so as to have complementary size, shape, charge and functional groups of the selected target by using the target molecule itself (such as a polypeptide, antibody, etc.), or a substance having a very similar structure, as its “print” or “template.” MIPs can be derivatized with the same reagents afforded to antibodies. For example, fluorescent ‘super’ MIPs can be coated onto beads or wells for use in highly sensitive separations or assays, or for use in high throughput screening of proteins.

Moreover, MIPs based upon the structure of the polypeptide(s) of the present invention may be useful in screening for compounds that bind to the polypeptide(s) of the invention. Such a MIP would serve the role of a synthetic “receptor” by minimicking the native architecture of the polypeptide. In fact, the ability of a MIP to serve the role of a synthetic receptor has already been demonstrated for the estrogen receptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760–5, (2001); Dickert, F, L., Hayden, O., Halikias, K, P, Analyst., 126(6):766–71, (2001)). A synthetic receptor may either be mimicked in its entirety (e.g., as the entire protein), or mimicked as a series of short peptides corresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys, Acta., 1544(1–2):255–66, (2001)). Such a synthetic receptor MIPs may be employed in any one or more of the screening methods described elsewhere herein.

MIPs have also been shown to be useful in “sensing” the presence of its mimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens, Bioelectron., 16(3):179–85, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798–802, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798–802, (2001)). For example, a MIP designed using a polypeptide of the present invention may be used in assays designed to identify, and potentially quantitate, the level of said polypeptide in a sample. Such a MIP may be used as a substitute for any component described in the assays, or kits, provided herein (e.g., ELISA, etc.).

A number of methods may be employed to create MIPs to a specific receptor, ligand, polypeptide, peptide, organic molecule. Several preferred methods are described by Esteban et al in J. Anal, Chem., 370(7):795–802, (2001), which is hereby incorporated herein by reference in its entirety in addition to any references cited therein. Additional methods are known in the art and are encompassed by the present invention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem, Soc., 123(9):2072–3, (2001); and Quaglia, M., Chenon, K., Hall, A, J., De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146–54, (2001); which are hereby incorporated by reference in their entirety herein.

Uses for Antibodies Directed Against Polypeptides of the Invention

The antibodies of the present invention have various utilities. For example, such antibodies may be used in diagnostic assays to detect the presence or quantification of the polypeptides of the invention in a sample. Such a diagnostic assay may be comprised of at least two steps. The first, subjecting a sample with the antibody, wherein the sample is a tissue (e.g., human, animal, etc.), biological fluid (e.g., blood, urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract (e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g., See Arenkov P, et al., Anal Biochem., 278(2):123–131 (2000)), or a chromatography column, etc. And a second step involving the quantification of antibody bound to the substrate. Alternatively, the method may additionally involve a first step of attaching the antibody, either covalently, electrostatically, or reversibly, to a solid support, and a second step of subjecting the bound antibody to the sample, as defined above and elsewhere herein.

Various diagnostic assay techniques are known in the art, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., (1987), pp147–158). The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase, green fluorescent protein, or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem., 13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219(1981); and Nygren, J. Histochem. And Cytochem., 30:407 (1982).

Antibodies directed against the polypeptides of the present invention are useful for the affinity purification of such polypeptides from recombinant cell culture or natural sources. In this process, the antibodies against a particular polypeptide are immobilized on a suitable support, such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the polypeptides to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except for the desired polypeptides, which are bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the desired polypeptide from the antibody.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. The translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737–49 (1999)).

These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1–4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%–20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses of Antibodies

The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, and 10−15 M.

Antibodies directed against polypeptides of the present invention are useful for inhibiting allergic reactions in animals. For example, by administering a therapeutically acceptable dose of an antibody, or antibodies, of the present invention, or a cocktail of the present antibodies, or in combination with other antibodies of varying sources, the animal may not elicit an allergic response to antigens.

Likewise, one could envision cloning the gene encoding an antibody directed against a polypeptide of the present invention, said polypeptide having the potential to elicit an allergic and/or immune response in an organism, and transforming the organism with said antibody gene such that it is expressed (e.g., constitutively, inducibly, etc.) in the organism. Thus, the organism would effectively become resistant to an allergic response resulting from the ingestion or presence of such an immune/allergic reactive polypeptide. Moreover, such a use of the antibodies of the present invention may have particular utility in preventing and/or ameliorating autoimmune diseases and/or disorders, as such conditions are typically a result of antibodies being directed against endogenous proteins. For example, in the instance where the polypeptide of the present invention is responsible for modulating the immune response to auto-antigens, transforming the organism and/or individual with a construct comprising any of the promoters disclosed herein or otherwise known in the art, in addition, to a polynucleotide encoding the antibody directed against the polypeptide of the present invention could effective inhibit the organisms immune system from eliciting an immune response to the auto-antigen(s). Detailed descriptions of therapeutic and/or gene therapy applications of the present invention are provided elsewhere herein.

Alternatively, antibodies of the present invention could be produced in a plant (e.g., cloning the gene of the antibody directed against a polypeptide of the present invention, and transforming a plant with a suitable vector comprising said gene for constitutive expression of the antibody within the plant), and the plant subsequently ingested by an animal, thereby conferring temporary immunity to the animal for the specific antigen the antibody is directed towards (See, for example, U.S. Pat. Nos. 5,914,123 and 6,034,298).

In another embodiment, antibodies of the present invention, preferably polyclonal antibodies, more preferably monoclonal antibodies, and most preferably single-chain antibodies, can be used as a means of inhibiting gene expression of a particular gene, or genes, in a human, mammal, and/or other organism. See, for example, International Publication Number WO 00/05391, published Feb. 3, 2000, to Dow Agrosciences LLC. The application of such methods for the antibodies of the present invention are known in the art, and are more particularly described elsewhere herein.

In yet another embodiment, antibodies of the present invention may be useful for multimerizing the polypeptides of the present invention. For example, certain proteins may confer enhanced biological activity when present in a multimeric state (i.e., such enhanced activity may be due to the increased effective concentration of such proteins whereby more protein is available in a localized location).

Antibody-based Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488–505 (1993); Wu and Wu, Biotherapy 3:87–95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573–596 (1993); Mulligan, Science 260:926–932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191–217 (1993); May, TIBTECH 11(5):155–215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932–8935 (1989); Zijlstra et al., Nature 342:435–438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429–4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932–8935 (1989); Zijlstra et al., Nature 342:435–438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581–599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291–302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644–651 (1994); Kiem et al., Blood 83:1467–1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129–141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110–114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499–503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3–10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431–434 (1991); Rosenfeld et al., Cell 68:143–155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225–234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775–783 (1995). In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289–300 (1993); U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599–618 (1993); Cohen et al., Meth. Enzymol. 217:618–644 (1993); Cline, Pharmac. Ther. 29:69–92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973–985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.

Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429–4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.

In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527–1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353–365 (1989); Lopez-Berestein, ibid., pp. 317–327; see generally ibid.)

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115–138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527–1533 (1990)).

In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864–1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging With Antibodies

Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976–985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087–3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

Kits

The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).

In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.

In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or calorimetric substrate (Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because certain proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Similarly, peptide cleavage sites can be introduced in-between such peptide moieties, which could additionally be subjected to protease activity to remove said peptide(s) from the protein of the present invention. The addition of peptide moieties, including peptide cleavage sites, to facilitate handling of polypeptides are familiar and routine techniques in the art.

Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84–86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958–3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of the constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52–58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459–9471 (1995).)

Moreover, the polypeptides of the present invention can be fused to marker sequences (also referred to as “tags”). Due to the availability of antibodies specific to such “tags”, purification of the fused polypeptide of the invention, and/or its identification is significantly facilitated since antibodies specific to the polypeptides of the invention are not required. Such purification may be in the form of an affinity purification whereby an anti-tag antibody or another type of affinity matrix (e.g., anti-tag antibody attached to the matrix of a flow-thru column) that binds to the epitope tag is present. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821–824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984)).

The skilled artisan would acknowledge the existence of other “tags” which could be readily substituted for the tags referred to supra for purification and/or identification of polypeptides of the present invention (Jones C., et al., J Chromatogr A. 707(1):3–22 (1995)). For example, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology 5:3610–3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547–553 (1990), the Flag-peptide—i.e., the octapeptide sequence DYKDDDDK (SEQ ID NO:28), (Hopp et al., Biotech. 6:1204–1210 (1988); the KT3 epitope peptide (Martin et al., Science, 255:192–194 (1992)); a-tubulin epitope peptide (Skinner et al., J. Biol. Chem., 266:15136–15166, (1991)); the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci. USA, 87:6363–6397 (1990)), the FITC epitope (Zymed, Inc.), the GFP epitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).

The present invention also encompasses the attachment of up to nine codons encoding a repeating series of up to nine arginine amino acids to the coding region of a polynucleotide of the present invention. The invention also encompasses chemically derivitizing a polypeptide of the present invention with a repeating series of up to nine arginine amino acids. Such a tag, when attached to a polypeptide, has recently been shown to serve as a universal pass, allowing compounds access to the interior of cells without additional derivitization or manipulation (Wender, P., et al., unpublished data).

Protein fusions involving polypeptides of the present invention, including fragments and/or variants thereof, can be used for the following, non-limiting examples, subcellular localization of proteins, determination of protein-protein interactions via immunoprecipitation, purification of proteins via affinity chromatography, functional and/or structural characterization of protein. The present invention also encompasses the application of hapten specific antibodies for any of the uses referenced above for epitope fusion proteins. For example, the polypeptides of the present invention could be chemically derivatized to attach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to the availability of monoclonal antibodies specific to such haptens, the protein could be readily purified using immunoprecipation, for example.

Polypeptides of the present invention, including fragments and/or variants thereof, in addition to, antibodies directed against such polypeptides, fragments, and/or variants, may be fused to any of a number of known, and yet to be determined, toxins, such as ricin, saporin (Mashiba H, et al., Ann. N.Y. Acad. Sci. 1999;886:233–5), or HC toxin (Tonukari N.J., et al., Plant Cell. February 2000;12(2):237–248), for example. Such fusions could be used to deliver the toxins to desired tissues for which a ligand or a protein capable of binding to the polypeptides of the invention exists.

The invention encompasses the fusion of antibodies directed against polypeptides of the present invention, including variants and fragments thereof, to said toxins for delivering the toxin to specific locations in a cell, to specific tissues, and/or to specific species. Such bifunctional antibodies are known in the art, though a review describing additional advantageous fusions, including citations for methods of production, can be found in P. J. Hudson, Curr. Opp. In. Imm. 11:548–557, (1999); this publication, in addition to the references cited therein, are hereby incorporated by reference in their entirety herein. In this context, the term “toxin” may be expanded to include any heterologous protein, a small molecule, radionucleotides, cytotoxic drugs, liposomes, adhesion molecules, glycoproteins, ligands, cell or tissue-specific ligands, enzymes, of bioactive agents, biological response modifiers, anti-fungal agents, hormones, steroids, vitamins, peptides, peptide analogs, anti-allergenic agents, anti-tubercular agents, anti-viral agents, antibiotics, anti-protozoan agents, chelates, radioactive particles, radioactive ions, X-ray contrast agents, monoclonal antibodies, polyclonal antibodies and genetic material. In view of the present disclosure, one skilled in the art could determine whether any particular “toxin” could be used in the compounds of the present invention. Examples of suitable “toxins” listed above are exemplary only and are not intended to limit the “toxins” that may be used in the present invention.

Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express the polypeptide of the present invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O2. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111–21 (1985); Koutz, P. J, et al., Yeast 5:167–77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 5:3859–76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a protein of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG, as required.

In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761, issued Mar. 31, 1998; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932–8935 (1989); and Zijlstra et al., Nature 342:435–438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105–111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein, the addition of epitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin, maltose binding protein, etc.), attachment of affinity tags such as biotin and/or streptavidin, the covalent attachment of chemical moieties to the amino acid backbone, N- or C-terminal processing of the polypeptides ends (e.g., proteolytic processing), deletion of the N-terminal methionine residue, etc.

Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The invention further encompasses chemical derivitization of the polypeptides of the present invention, preferably where the chemical is a hydrophilic polymer residue. Exemplary hydrophilic polymers, including derivatives, may be those that include polymers in which the repeating units contain one or more hydroxy groups (polyhydroxy polymers), including, for example, poly(vinyl alcohol); polymers in which the repeating units contain one or more amino groups (polyamine polymers), including, for example, peptides, polypeptides, proteins and lipoproteins, such as albumin and natural lipoproteins; polymers in which the repeating units contain one or more carboxy groups (polycarboxy polymers), including, for example, carboxymethylcellulose, alginic acid and salts thereof, such as sodium and calcium alginate, glycosaminoglycans and salts thereof, including salts of hyaluronic acid, phosphorylated and sulfonated derivatives of carbohydrates, genetic material, such as interleukin-2 and interferon, and phosphorothioate oligomers; and polymers in which the repeating units contain one or more saccharide moieties (polysaccharide polymers), including, for example, carbohydrates.

The molecular weight of the hydrophilic polymers may vary, and is generally about 50 to about 5,000,000, with polymers having a molecular weight of about 100 to about 50,000 being preferred. The polymers may be branched or unbranched. More preferred polymers have a molecular weight of about 150 to about 10,000, with molecular weights of 200 to about 8,000 being even more preferred.

For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

Additional preferred polymers which may be used to derivatize polypeptides of the invention, include, for example, poly(ethylene glycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate and poly(vinyl alcohol), with PEG polymers being particularly preferred. Preferred among the PEG polymers are PEG polymers having a molecular weight of from about 100 to about 10,000. More preferably, the PEG polymers have a molecular weight of from about 200 to about 8,000, with PEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of 2,000, 5,000 and 8,000, respectively, being even more preferred. Other suitable hydrophilic polymers, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, the polymers used may include polymers that can be attached to the polypeptides of the invention via alkylation or acylation reactions.

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028–1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminus) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As with the various polymers exemplified above, it is contemplated that the polymeric residues may contain functional groups in addition, for example, to those typically involved in linking the polymeric residues to the polypeptides of the present invention. Such functionalities include, for example, carboxyl, amine, hydroxy and thiol groups. These functional groups on the polymeric residues can be further reacted, if desired, with materials that are generally reactive with such functional groups and which can assist in targeting specific tissues in the body including, for example, diseased tissue. Exemplary materials which can be reacted with the additional functional groups include, for example, proteins, including antibodies, carbohydrates, peptides, glycopeptides, glycolipids, lectins, and nucleosides.

In addition to residues of hydrophilic polymers, the chemical used to derivatize the polypeptides of the present invention can be a saccharide residue. Exemplary saccharides which can be derived include, for example, monosaccharides or sugar alcohols, such as erythrose, threose, ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitol and sedoheptulose, with preferred monosaccharides being fructose, mannose, xylose, arabinose, mannitol and sorbitol; and disaccharides, such as lactose, sucrose, maltose and cellobiose. Other saccharides include, for example, inositol and ganglioside head groups. Other suitable saccharides, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, saccharides which may be used for derivitization include saccharides that can be attached to the polypeptides of the invention via alkylation or acylation reactions.

Moreover, the invention also encompasses derivitization of the polypeptides of the present invention, for example, with lipids (including cationic, anionic, polymerized, charged, synthetic, saturated, unsaturated, and any combination of the above, etc.). stabilizing agents.

The invention encompasses derivitization of the polypeptides of the present invention, for example, with compounds that may serve a stabilizing function (e.g., to increase the polypeptides half-life in solution, to make the polypeptides more water soluble, to increase the polypeptides hydrophilic or hydrophobic character, etc.). Polymers useful as stabilizing materials may be of natural, semi-synthetic (modified natural) or synthetic origin. Exemplary natural polymers include naturally occurring polysaccharides, such as, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch and various other natural homopolymer or heteropolymers, such as those containing one or more of the following aldoses, ketoses, acids or amines: erythose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, and naturally occurring derivatives thereof Accordingly, suitable polymers include, for example, proteins, such as albumin, polyalginates, and polylactide-coglycolide polymers. Exemplary semi-synthetic polymers include carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose. Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes (such as, for example, polyethylene glycol (including for example, the class of compounds referred to as Pluronics.RTM., commercially available from BASF, Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate), polypropylenes (such as, for example, polypropylene glycol), polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinyl chloride and polyvinylpyrrolidone), polyamides including nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers (such as, for example, polytetrafluoroethylene), acrylate, methacrylate, and polymethylmethacrylate, and derivatives thereof. Methods for the preparation of derivatized polypeptides of the invention which employ polymers as stabilizing compounds will be readily apparent to one skilled in the art, in view of the present disclosure, when coupled with information known in the art, such as that described and referred to in Unger, U.S. Pat. No. 5,205,290, the disclosure of which is hereby incorporated by reference herein in its entirety.

Moreover, the invention encompasses additional modifications of the polypeptides of the present invention. Such additional modifications are known in the art, and are specifically provided, in addition to methods of derivitization, etc., in U.S. Pat. No. 6,028,066, which is hereby incorporated in its entirety herein.

The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO:2 or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides as described herein). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.

In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.

Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.

In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence. In a further embodiment, associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.

The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

In addition, the polynucleotide insert of the present invention could be operatively linked to “artificial” or chimeric promoters and transcription factors. Specifically, the artificial promoter could comprise, or alternatively consist, of any combination of cis-acting DNA sequence elements that are recognized by trans-acting transcription factors. Preferably, the cis acting DNA sequence elements and trans-acting transcription factors are operable in mammals. Further, the trans-acting transcription factors of such “artificial” promoters could also be “artificial” or chimeric in design themselves and could act as activators or repressors to said “artificial” promoter.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15–25 bp) from the sequences shown in SEQ ID NO:1. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO:1 will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000–4,000 bp are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. Disease mapping data are known in the art. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50–500 potential causative genes.

Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected organisms can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected organisms, but not in normal organisms, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal organisms is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affected organisms as compared to unaffected organisms can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an organism and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.

By “measuring the expression level of a polynucleotide of the present invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the present invention or the level of the mRNA encoding the polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of organisms not having a disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained from an organism, body fluids, cell line, tissue culture, or other source which contains the polypeptide of the present invention or mRNA. As indicated, biological samples include body fluids (such as the following non-limiting examples, sputum, amniotic fluid, urine, saliva, breast milk, secretions, interstitial fluid, blood, serum, spinal fluid, etc.) which contain the polypeptide of the present invention, and other tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from organisms are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

The method(s) provided above may Preferably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support. In one exemplary method, the support may be a “gene chip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the present invention attached may be used to identify polymorphisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, including proliferative diseases and conditions. Such a method is described in U.S. Pat. Nos. 5,858,659 and 5,856,104. The U.S. Patents referenced supra are hereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the stronger binding characteristics of PNA:DNA hybrids. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°–20° C., vs. 4°–16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.

In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991) ) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat or prevent disease.

The present invention encompasses the addition of a nuclear localization signal, operably linked to the 5′ end, 3′ end, or any location therein, to any of the oligonucleotides, anti sense oligonucleotides, triple helix oligonucleotides, ribozymes, PNA oligonucleotides, and/or polynucleotides, of the present invention. See, for example, G. Cutrona, et al., Nat. Biotech., 18:300–303, (2000); which is hereby incorporated herein by reference.

Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. In one example, polynucleotide sequences of the present invention may be used to construct chimeric RNA/DNA oligonucleotides corresponding to said sequences, specifically designed to induce host cell mismatch repair mechanisms in an organism upon systemic injection, for example (Bartlett, R. J., et al., Nat. Biotech, 18:615–622 (2000), which is hereby incorporated by reference herein in its entirety). Such RNA/DNA oligonucleotides could be designed to correct genetic defects in certain host strains, and/or to introduce desired phenotypes in the host (e.g., introduction of a specific polymorphism within an endogenous gene corresponding to a polynucleotide of the present invention that may ameliorate and/or prevent a disease symptom and/or disorder, etc.). Alternatively, the polynucleotide sequence of the present invention may be used to construct duplex oligonucleotides corresponding to said sequence, specifically designed to correct genetic defects in certain host strains, and/or to introduce desired phenotypes into the host (e.g., introduction of a specific polymorphism within an endogenous gene corresponding to a polynucleotide of the present invention that may ameliorate and/or prevent a disease symptom and/or disorder, etc). Such methods of using duplex oligonucleotides are known in the art and are encompassed by the present invention (see EP1007712, which is hereby incorporated by reference herein in its entirety).

The polynucleotides are also useful for identifying organisms from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an organisms genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, organisms can be identified because each organism will have a unique set of DNA sequences. Once an unique ID database is established for an organism, positive identification of that organism, living or dead, can be made from extremely small tissue samples. Similarly, polynucleotides of the present invention can be used as polymorphic markers, in addition to, the identification of transformed or non-transformed cells and/or tissues.

There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination. Moreover, as mentioned above, such reagents can be used to screen and/or identify transformed and non-transformed cells and/or tissues.

In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.

A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al., J. Cell. Biol. 101:976–985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087–3096 (1987).) Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (125I, 121I ), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Moreover, polypeptides of the present invention can be used to treat, prevent, and/or diagnose disease. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor suppressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).

Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat, prevent, and/or diagnose disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of a polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the invention that operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207–216 (1993); Ferrantini et al., Cancer Research, 53:107–1112 (1993); Ferrantini et al., J. Immunology 153: 4604–4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221–229 (1995); Ogura et al., Cancer Research 50: 5102–5106 (1990); Santodonato, et al., Human Gene Therapy 7:1–10 (1996); Santodonato, et al., Gene Therapy 4:1246–1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31–38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.

As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

In one embodiment, the polynucleotide of the invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.

The polynucleotide vector constructs of the invention used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used for driving the expression of polynucleotide sequence of the invention. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the polynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct of the invention can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/lkg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.

In certain embodiments, the polynucleotide constructs of the invention are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA , 84:7413–7416 (1987), which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA , 86:6077–6081 (1989), which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem., 265:10189–10192 (1990), which is herein incorporated by reference), in functional form.

Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA , 84:7413–7416 (1987), which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication NO: WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413–7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology, 101:512–527 (1983), which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca2+-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilson et al., Cell , 17:77 (1979)); ether injection (Deamer et al., Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad. Sci. USA , 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl. Acad. Sci. USA , 75:145 (1978); Schaefer-Ridder et al., Science, 215:166 (1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide methods for delivering DNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding polypeptides of the invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm 12, and DAN cell lines as described in Miller, Human Gene Therapy, 1:5–14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides of the invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express polypeptides of the invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotides of the invention contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses polypeptides of the invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir. Dis., 109:233–238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld et al., Science, 252:431–434 (1991); Rosenfeld et al., Cell, 68:143–155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA, 76:6606 (1979)).

Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499–503 (1993); Rosenfeld et al., Cell , 68:143–155 (1992); Engelhardt et al., Human Genet. Ther., 4:759–769 (1993); Yang et al., Nature Genet., 7:362–369 (1994); Wilson et al., Nature, 365:691–692 (1993); and U.S. Pat. No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol., 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct containing polynucleotides of the invention is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct of the invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express the desired gene product.

Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding the polypeptide sequence of interest) via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932–8935 (1989); and Zijistra et al., Nature, 342:435–438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.

The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.

The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.

The polynucleotides encoding polypeptides of the present invention may be administered along with other polynucleotides encoding angiogenic proteins. Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.

Preferably, the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers. (Kaneda et al., Science, 243:375 (1989)).

A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA, 189:11277–11281 (1992), which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.

Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian. Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.

Biological Activities

The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.

Immune Activity

The polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer or some autoimmune diseases, disorders, and/or conditions, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells. A polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein diseases, disorders, and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, arterial thrombosis, venous thrombosis, etc.), blood platelet diseases, disorders, and/or conditions (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. Polynucleotides or polypeptides, or agonists or antagonists of the present invention are may also be useful for the detection, prognosis, treatment, and/or prevention of heart attacks (infarction), strokes, scarring, fibrinolysis, uncontrolled bleeding, uncontrolled coagulation, uncontrolled complement fixation, and/or inflammation.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be useful in treating, preventing, and/or diagnosing autoimmune diseases, disorders, and/or conditions. Many autoimmune diseases, disorders, and/or conditions result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune diseases, disorders, and/or conditions.

Examples of autoimmune diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed or detected by the present invention include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyclitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or agonists or antagonists of the present invention. Moreover, these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to treat, prevent, and/or diagnose organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.

Similarly, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to modulate inflammation. For example, the polypeptide or polynucleotide or agonists or antagonist may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat, prevent, and/or diagnose inflammatory conditions, both chronic and acute conditions, including chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)

Hyperproliferative Disorders

A polynucleotides or polypeptides, or agonists or antagonists of the invention can be used to treat, prevent, and/or diagnose hyperproliferative diseases, disorders, and/or conditions, including neoplasms. A polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative diseases, disorders, and/or conditions can be treated, prevented, and/or diagnosed. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating, preventing, and/or diagnosing hyperproliferative diseases, disorders, and/or conditions, such as a chemotherapeutic agent.

Examples of hyperproliferative diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

Similarly, other hyperproliferative diseases, disorders, and/or conditions can also be treated, prevented, and/or diagnosed by a polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative diseases, disorders, and/or conditions include, but are not limited to: hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/or conditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

One preferred embodiment utilizes polynucleotides of the present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating or preventing cell proliferative diseases, disorders, and/or conditions by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method of treating or preventing cell-proliferative diseases, disorders, and/or conditions in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the polynucleotides of the present invention is inserted into cells to be treated utilizing a retrovirus, or more Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324–326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By “repressing expression of the oncogenic genes” is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.

For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.

Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By “biologically inhibiting” is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating, preventing, and/or diagnosing one or more of the described diseases, disorders, and/or conditions. Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the present invention are useful for treating, preventing, and/or diagnosing a subject having or developing cell proliferative and/or differentiation diseases, disorders, and/or conditions as described herein. Such treatment comprises administering a single or multiple doses of the antibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of diseases, disorders, and/or conditions related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10−6M, 10−6M, 5×10−7M, 10−7M, 5×10−8M, 10−8M, 5×10−9M, 10−9M, 5×10−10M, 10−10M, 5×10−11M, 10−11M, 5×10−12M, 10−12M, 5×10−13M, 10−13M, 5×10−14M, 10−14M, 5×10−15M, and 10−15M.

Moreover, polypeptides of the present invention may be useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648–53 (1998), which is hereby incorporated by reference). Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155–61 (1998), which is hereby incorporated by reference)).

Polypeptides, including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et al., Eur J Biochem 254(3):439–59 (1998), which is hereby incorporated by reference). Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins, antiinflammatory proteins (See for example, Mutat. Res. 400(1–2):447–55 (1998), Med Hypotheses. 50(5):423–33 (1998), Chem. Biol. Interact. Apr 24;111–112:23–34 (1998), J Mol Med. 76(6):402–12 (1998), Int. J. Tissue React. 20(1):3–15 (1998), which are all hereby incorporated by reference).

Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewhere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125–41, which is hereby incorporated by reference). Such therapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention. Polypeptides or polypeptide antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention ‘vaccinated’ the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.

Cardiovascular Disorders

Polynucleotides or polypeptides, or agonists or antagonists of the invention may be used to treat, prevent, and/or diagnose cardiovascular diseases, disorders, and/or conditions, including peripheral artery disease, such as limb ischemia.

Cardiovascular diseases, disorders, and/or conditions include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.

Cardiovascular diseases, disorders, and/or conditions also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders, and/or conditions, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.

Cerebrovascular diseases, disorders, and/or conditions include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.

Polynucleotides or polypeptides, or agonists or antagonists of the invention, are especially effective for the treatment of critical limb ischemia and coronary disease.

Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides of the invention may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides of the invention are described in more detail herein.

Anti-angiogenesis Activity

The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., Cell 56:345–355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye diseases, disorders, and/or conditions, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630–634 (1991); Folkman et al., N. Engl. J. Med., 333:1757–1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401–411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175–203 (1985); Patz, Am. J. Opthalmol. 94:715–743 (1982); and Folkman et al., Science 221:719–725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442–447 (1987).

The present invention provides for treatment of diseases, disorders, and/or conditions associated with neovascularization by administration of the polynucleotides and/or polypeptides of the invention, as well as agonists or antagonists of the present invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)). Thus, the present invention provides a method of treating, preventing, and/or diagnosing an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the invention. For example, polynucleotides, polypeptides, antagonists and/or agonists may be utilized in a variety of additional methods in order to therapeutically treat or prevent a cancer or tumor. Cancers which may be treated, prevented, and/or diagnosed with polynucleotides, polypeptides, antagonists and/or agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat or prevent cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

Within yet other aspects, polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Polynucleotides, polypeptides, antagonists and/or agonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and/or agonists may be useful in treating, preventing, and/or diagnosing other diseases, disorders, and/or conditions, besides cancers, which involve angiogenesis. These diseases, disorders, and/or conditions include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scieroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, within one aspect of the present invention methods are provided for treating, preventing, and/or diagnosing hypertrophic scars and keloids, comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist of the invention to a hypertrophic scar or keloid.

Within one embodiment of the present invention polynucleotides, polypeptides, antagonists and/or agonists are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating, preventing, and/or diagnosing neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.

Moreover, Ocular diseases, disorders, and/or conditions associated with neovascularization which can be treated, prevented, and/or diagnosed with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal. 85:704–710 (1978) and Gartner et al., Surv. Ophthal. 22:291–312 (1978).

Thus, within one aspect of the present invention methods are provided for treating or preventing neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (as described above) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood vessels. In certain pathological conditions however, capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of diseases, disorders, and/or conditions can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.

Within particularly preferred embodiments of the invention, may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.

Within other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to “protect” the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2–3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.

Within another aspect of the present invention, methods are provided for treating or preventing neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat or prevent early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Within other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating or preventing proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided for treating or preventing retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.

Additionally, diseases, disorders, and/or conditions which can be treated, prevented, and/or diagnosed with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

Moreover, diseases, disorders, and/or conditions and/or states, which can be treated, prevented, and/or diagnosed with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a “morning after” method. Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.

Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be incorporated into surgical sutures in order to prevent stitch granulomas.

Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.

Within one aspect of the present invention, polynucleotides, polypeptides, agonists and/or agonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.

The polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.

Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22–26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321–17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475–480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555–557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440–1446, 1987); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659–1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; Takeuchi et al., Agents Actions 36:312–316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated, prevented, and/or diagnosed by the polynucleotides or polypeptides and/or antagonists or agonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune diseases, disorders, and/or conditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection. In preferred embodiments, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cell survival that could be treated, prevented or diagnosed by the polynucleotides or polypeptides, or agonists or antagonists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis that could be treated, prevented, and/or diagnosed by the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, include AIDS; neurodegenerative diseases, disorders, and/or conditions (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune diseases, disorders, and/or conditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or antagonists of the invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote dermal reestablishment subsequent to dermal loss

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are a non-exhaustive list of grafts that polynucleotides or polypeptides, agonists or antagonists of the invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepidermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, can be used to promote skin strength and to improve the appearance of aged skin.

It is believed that the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may have a cytoprotective effect on the small intestine mucosa. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat diseases associate with the under expression of the polynucleotides of the invention.

Moreover, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to prevent and heal damage to the lungs due to various pathological states. A growth factor such as the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated, prevented, and/or diagnosed using the polynucleotides or polypeptides, and/or agonists or antagonists of the invention. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).

In addition, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.

Neurological Diseases

Nervous system diseases, disorders, and/or conditions, which can be treated, prevented, and/or diagnosed with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases, disorders, and/or conditions which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated, prevented, and/or diagnosed in a patient (including human and non-human mammalian patients) according to the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases, disorders, and/or conditions, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

In a preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral hypoxia. In one aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral ischemia. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral infarction. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose or prevent neural cell injury associated with a stroke. In a further aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with a heart attack.

The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, the method set forth in Arakawa et al. (J. Neurosci. 10:3507–3515 (1990)); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al. (Exp. Neurol. 70:65–82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17–42 (1981)); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron diseases, disorders, and/or conditions that may be treated, prevented, and/or diagnosed according to the invention include, but are not limited to, diseases, disorders, and/or conditions such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as diseases, disorders, and/or conditions that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Infectious Disease

A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to treat, prevent, and/or diagnose infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated, prevented, and/or diagnosed. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polypeptide or polynucleotide and/or agonist or antagonist of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Bimaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: tetanus, Diptheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used totreat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose malaria.

Preferably, treatment or prevention using a polypeptide or polynucleotide and/or agonist or antagonist of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.

Regeneration

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59–87 (1997).) The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated, prevented, and/or diagnosed include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide and/or agonist or antagonist of the present invention to proliferate and differentiate nerve cells. Diseases that could be treated, prevented, and/or diagnosed using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic diseases, disorders, and/or conditions (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated, prevented, and/or diagnosed using the polynucleotide or polypeptide and/or agonist or antagonist of the present invention.

Chemotaxis

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat, prevent, and/or diagnose inflammation, infection, hyperproliferative diseases, disorders, and/or conditions, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat, prevent, and/or diagnose wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat, prevent, and/or diagnose wounds.

It is also contemplated that a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may inhibit chemotactic activity. These molecules could also be used to treat, prevent, and/or diagnose diseases, disorders, and/or conditions. Thus, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention could be used as an inhibitor of chemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors),or small molecules.

Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.

Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.

Additionally, the receptor to which a polypeptide of the invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labeled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.

As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.

Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of polypeptides of the invention thereby effectively generating agonists and antagonists of polypeptides of the invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724–33 (1997); Harayama, S. Trends Biotechnol. 16(2):76–82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265–76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308–13 (1998) (each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides and corresponding polypeptides of the invention may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired polynucleotide sequence of the invention molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides and corresponding polypeptides of the invention may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptides of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of the polypeptides of the invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and 3[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3[H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3[H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat, prevent, and/or diagnose disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues. Therefore, the invention includes a method of identifying compounds which bind to the polypeptides of the invention comprising the steps of: (a) incubating a candidate binding compound with the polypeptide; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with the polypeptide, (b) assaying a biological activity, and (b) determining if a biological activity of the polypeptide has been altered.

Also, one could identify molecules bind a polypeptide of the invention experimentally by using the beta-pleated sheet regions contained in the polypeptide sequence of the protein. Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of each beta pleated sheet regions in a disclosed polypeptide sequence. Additional embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, any combination or all of contained in the polypeptide sequences of the invention. Additional preferred embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, the amino acid sequence of each of the beta pleated sheet regions in one of the polypeptide sequences of the invention. Additional embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, any combination or all of the beta pleated sheet regions in one of the polypeptide sequences of the invention.

Targeted Delivery

In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention.

As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.

This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention. These methods comprise contacting such an agent with a polypeptide of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the present invention.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.

The human MMP-29 polypeptides and/or peptides of the present invention, or immunogenic fragments or oligopeptides thereof, can be used for screening therapeutic drugs or compounds in a variety of drug screening techniques. The fragment employed in such a screening assay may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The reduction or abolition of activity of the formation of binding complexes between the ion channel protein and the agent being tested can be measured. Thus, the present invention provides a method for screening or assessing a plurality of compounds for their specific binding affinity with a MMP-29 polypeptide, or a bindable peptide fragment, of this invention, comprising providing a plurality of compounds, combining the MMP-29 polypeptide, or a bindable peptide fragment, with each of a plurality of compounds for a time sufficient to allow binding under suitable conditions and detecting binding of the MMP-29 polypeptide or peptide to each of the plurality of test compounds, thereby identifying the compounds that specifically bind to the MMP-29 polypeptide or peptide.

Methods of identifying compounds that modulate the activity of the novel human MMP-29 polypeptides and/or peptides are provided by the present invention and comprise combining a potential or candidate compound or drug modulator of metalloprotease biological activity with an MMP-29 polypeptide or peptide, for example, the MMP-29 amino acid sequence as set forth in SEQ ID NO:2, and measuring an effect of the candidate compound or drug modulator on the biological activity of the MMP-29 polypeptide or peptide. Such measurable effects include, for example, physical binding interaction; the ability to cleave a suitable metalloprotease substrate; effects on native and cloned MMP-29-expressing cell line; and effects of modulators or other metalloprotease-mediated physiological measures.

Another method of identifying compounds that modulate the biological activity of the novel MMP-29 polypeptides of the present invention comprises combining a potential or candidate compound or drug modulator of a metalloprotease biological activity with a host cell that expresses the MMP-29 polypeptide and measuring an effect of the candidate compound or drug modulator on the biological activity of the MMP-29 polypeptide. The host cell can also be capable of being induced to express the MMP-29 polypeptide, e.g., via inducible expression. Physiological effects of a given modulator candidate on the MMP-29 polypeptide can also be measured. Thus, cellular assays for particular metalloprotease modulators may be either direct measurement or quantification of the physical biological activity of the MMP-29 polypeptide, or they may be measurement or quantification of a physiological effect. Such methods preferably employ a MMP-29 polypeptide as described herein, or an overexpressed recombinant MMP-29 polypeptide in suitable host cells containing an expression vector as described herein, wherein the MMP-29 polypeptide is expressed, overexpressed, or undergoes upregulated expression.

Another aspect of the present invention embraces a method of screening for a compound that is capable of modulating the biological activity of a MMP-29 polypeptide, comprising providing a host cell containing an expression vector harboring a nucleic acid sequence encoding a MMP-29 polypeptide, or a functional peptide or portion thereof (e.g., SEQ ID NOS: 2); determining the biological activity of the expressed MMP-29 polypeptide in the absence of a modulator compound; contacting the cell with the modulator compound and determining the biological activity of the expressed MMP-29 polypeptide in the presence of the modulator compound. In such a method, a difference between the activity of the MMP-29 polypeptide in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.

Essentially any chemical compound can be employed as a potential modulator or ligand in the assays according to the present invention. Compounds tested as metalloprotease modulators can be any small chemical compound, or biological entity (e.g., protein, sugar, nucleic acid, lipid). Test compounds will typically be small chemical molecules and peptides. Generally, the compounds used as potential modulators can be dissolved in aqueous or organic (e.g., DMSO-based) solutions. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source. Assays are typically run in parallel, for example, in microtiter formats on microtiter plates in robotic assays. There are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland), for example. Also, compounds may be synthesized by methods known in the art.

High throughput screening methodologies are particularly envisioned for the detection of modulators of the novel MMP-29 polynucleotides and polypeptides described herein. Such high throughput screening methods typically involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (e.g., ligand or modulator compounds). Such combinatorial chemical libraries or ligand libraries are then screened in one or more assays to identify those library members (e.g., particular chemical species or subclasses) that display a desired characteristic activity. The compounds so identified can serve as conventional lead compounds, or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemical compounds generated either by chemical synthesis or biological synthesis, by combining a number of chemical building blocks (i.e., reagents such as amino acids). As an example, a linear combinatorial library, e.g., a polypeptide or peptide library, is formed by combining a set of chemical building blocks in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide or peptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries is well known to those having skill in the pertinent art. Combinatorial libraries include, without limitation, peptide libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487–493; and Houghton et al., 1991, Nature, 354:84–88). Other chemistries for generating chemical diversity libraries can also be used. Nonlimiting examples of chemical diversity library chemistries include, peptides (PCT Publication No. WO 91/019735), encoded peptides (PCT Publication No. WO 93/20242), random bio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc. Natl. Acad. Sci. USA, 90:6909–6913), vinylogous polypeptides (Hagihara et al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J. Amer. Chem. Soc., 114:9217–9218), analogous organic synthesis of small compound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 16:2661), oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidyl phosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries (e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309–314) and PCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996, Science, 274–1520–1522) and U.S. Pat. No. 5,593,853), small organic molecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; and the like).

Devices for the preparation of combinatorial libraries are commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a large number of combinatorial libraries are commercially available (e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., and the like).

In one embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the cell or tissue expressing an ion channel is attached to a solid phase substrate. In such high throughput assays, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to perform a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5–10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 96 modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; thus, for example, assay screens for up to about 6,000–20,000 different compounds are possible using the described integrated systems.

In another of its aspects, the present invention encompasses screening and small molecule (e.g., drug) detection assays which involve the detection or identification of small molecules that can bind to a given protein, i.e., a MMP-29 polypeptide or peptide. Particularly preferred are assays suitable for high throughput screening methodologies.

In such binding-based detection, identification, or screening assays, a functional assay is not typically required. All that is needed is a target protein, preferably substantially purified, and a library or panel of compounds (e.g., ligands, drugs, small molecules) or biological entities to be screened or assayed for binding to the protein target. Preferably, most small molecules that bind to the target protein will modulate activity in some manner, due to preferential, higher affinity binding to functional areas or sites on the protein.

An example of such an assay is the fluorescence based thermal shift assay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) as described in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano et al.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assay allows the detection of small molecules (e.g., drugs, ligands) that bind to expressed, and preferably purified, ion channel polypeptide based on affinity of binding determinations by analyzing thermal unfolding curves of protein-drug or ligand complexes. The drugs or binding molecules determined by this technique can be further assayed, if desired, by methods, such as those described herein, to determine if the molecules affect or modulate function or activity of the target protein.

To purify a MMP-29 polypeptide or peptide to measure a biological binding or ligand binding activity, the source may be a whole cell lysate that can be prepared by successive freeze-thaw cycles (e.g., one to three) in the presence of standard protease inhibitors. The MMP-29 polypeptide may be partially or completely purified by standard protein purification methods, e.g., affinity chromatography using specific antibody described infra, or by ligands specific for an epitope tag engineered into the recombinant MMP-29 polypeptide molecule, also as described herein. Binding activity can then be measured as described.

Compounds which are identified according to the methods provided herein, and which modulate or regulate the biological activity or physiology of the MMP-29 polypeptides according to the present invention are a preferred embodiment of this invention. It is contemplated that such modulatory compounds may be employed in treatment and therapeutic methods for treating a condition that is mediated by the novel MMP-29 polypeptides by administering to an individual in need of such treatment a therapeutically effective amount of the compound identified by the methods described herein.

In addition, the present invention provides methods for treating an individual in need of such treatment for a disease, disorder, or condition that is mediated by the MMP-29 polypeptides of the invention, comprising administering to the individual a therapeutically effective amount of the MMP-29-modulating compound identified by a method provided herein.

Antisense and Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:1, or the complementary strand thereof, and/or to nucleotide sequences contained a deposited clone. In one embodiment, antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research, 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoR1 site on the 5 end and a HindIII site on the 3 end. Next, the pair of oligonucleotides is heated at 90° C. for one minute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5, 10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding a polypeptide of the invention, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature, 29:304–310 (1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22:787–797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441–1445 (1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296:39–42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids of the invention, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature, 372:333–335 (1994). Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of a polynucleotide sequence of the invention could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5′-, 3′- or coding region of mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553–6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648–652 (1987); PCT Publication NO: WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., BioTechniques, 6:958–976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539–549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., Nucl. Acids Res., 15:6625–6641 (1987)). The oligonucleotide is a 2-O-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131–6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327–330 (1987)).

Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A., 85:7448–7451 (1988)), etc.

While antisense nucleotides complementary to the coding region sequence of the invention could be used, those complementary to the transcribed untranslated region are most preferred.

Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al, Science, 247:1222–1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs corresponding to the polynucleotides of the invention, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585–591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within each nucleotide sequence disclosed in the sequence listing. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the mRNA corresponding to the polynucleotides of the invention; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the polynucleotides of the invention in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.

The antagonist/agonist may also be employed to treat, prevent, and/or diagnose the diseases described herein.

Thus, the invention provides a method of treating or preventing diseases, disorders, and/or conditions, including but not limited to the diseases, disorders, and/or conditions listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention. invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.

Biotic Associations

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations with other organisms. Such associations may be symbiotic, nonsymbiotic, endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature. In general, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability to form biotic associations with any member of the fungal, bacterial, lichen, mycorrhizal, cyanobacterial, dinoflaggellate, and/or algal, kingdom, phylums, families, classes, genuses, and/or species.

The mechanism by which a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the host organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations is variable, though may include, modulating osmolarity to desirable levels for the symbiont, modulating pH to desirable levels for the symbiont, modulating secretions of organic acids, modulating the secretion of specific proteins, phenolic compounds, nutrients, or the increased expression of a protein required for host-biotic organisms interactions (e.g., a receptor, ligand, etc.). Additional mechanisms are known in the art and are encompassed by the invention (see, for example, “Microbial Signalling and Communication”, eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, Cambridge University Press, Cambridge, (1999); which is hereby incorporated herein by reference).

In an alternative embodiment, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may decrease the host organisms ability to form biotic associations with another organism, either directly or indirectly. The mechanism by which a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may decrease the host organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations with another organism is variable, though may include, modulating osmolarity to undesirable levels, modulating pH to undesirable levels, modulating secretions of organic acids, modulating the secretion of specific proteins, phenolic compounds, nutrients, or the decreased expression of a protein required for host-biotic organisms interactions (e.g., a receptor, ligand, etc.). Additional mechanisms are known in the art and are encompassed by the invention (see, for example, “Microbial Signalling and Communication”, eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, Cambridge University Press, Cambridge, (1999); which is hereby incorporated herein by reference).

The hosts ability to maintain biotic associations with a particular pathogen has significant implications for the overall health and fitness of the host. For example, human hosts have symbiosis with enteric bacteria in their gastrointestinal tracts, particularly in the small and large intestine. In fact, bacteria counts in feces of the distal colon often approach 10¹² per milliliter of feces. Examples of bowel flora in the gastrointestinal tract are members of the Enterobacteriaceae, Bacteriodes, in addition to a-hemolytic streptococci, E. coli, Bifobacteria, Anaerobic cocci, Eubacteria, Costridia, lactobacilli, and yeasts. Such bacteria, among other things, assist the host in the assimilation of nutrients by breaking down food stuffs not typically broken down by the hosts digestive system, particularly in the hosts bowel. Therefore, increasing the hosts ability to maintain such a biotic association would help assure proper nutrition for the host.

Aberrations in the enteric bacterial population of mammals, particularly humans, has been associated with the following disorders: diarrhea, ileus, chronic inflammatory disease, bowel obstruction, duodenal diverticula, biliary calculous disease, and malnutrition. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention are useful for treating, detecting, diagnosing, prognosing, and/or ameliorating, either directly or indirectly, and of the above mentioned diseases and/or disorders associated with aberrant enteric flora population.

The composition of the intestinal flora, for example, is based upon a variety of factors, which include, but are not limited to, the age, race, diet, malnutrition, gastric acidity, bile salt excretion, gut motility, and immune mechanisms. As a result, the polynucleotides and polypeptides, including agonists, antagonists, and fragments thereof, may modulate the ability of a host to form biotic associations by affecting, directly or indirectly, at least one or more of these factors.

Although the predominate intestinal flora comprises anaerobic organisms, an underlying percentage represents aerobes (e.g., E. coli). This is significant as such aerobes rapidly become the predominate organisms in intraabdominal infections—effectively becoming opportunistic early in infection pathogenesis. As a result, there is an intrinsic need to control aerobe populations, particularly for immune compromised individuals.

In a preferred embodiment, a polynucleotides and polypeptides, including agonists, antagonists, and fragments thereof, are useful for inhibiting biotic associations with specific enteric symbiont organisms in an effort to control the population of such organisms.

Biotic associations occur not only in the gastrointestinal tract, but also on an in the integument. As opposed to the gastrointestinal flora, the cutaneous flora is comprised almost equally with aerobic and anaerobic organisms. Examples of cutaneous flora are members of the gram-positive cocci (e.g., S. aureus, coagulase-negative staphylococci, micrococcus, M. sedentarius), gram-positive bacilli (e.g., Corynebacterium species, C. minutissimum, Brevibacterium species, Propoionibacterium species, P. acnes), gram-negative bacilli (e.g., Acinebacter species), and fungi (Pityrosporum orbiculare). The relatively low number of flora associated with the integument is based upon the inability of many organisms to adhere to the skin. The organisms referenced above have acquired this unique ability. Therefore, the polynucleotides and polypeptides of the present invention may have uses which include modulating the population of the cutaneous flora, either directly or indirectly.

Aberrations in the cutaneous flora are associated with a number of significant diseases and/or disorders, which include, but are not limited to the following: impetigo, ecthyma, blistering distal dactulitis, pustules, folliculitis, cutaneous abscesses, pitted keratolysis, trichomycosis axcillaris, dermatophytosis complex, axillary odor, erthyrasma, cheesy foot odor, acne, tinea versicolor, seborrheic dermititis, and Pityrosporum folliculitis, to name a few. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention are useful for treating, detecting, diagnosing, prognosing, and/or ameliorating, either directly or indirectly, and of the above mentioned diseases and/or disorders associated with aberrant cutaneous flora population.

Additional biotic associations, including diseases and disorders associated with the aberrant growth of such associations, are known in the art and are encompassed by the invention. See, for example, “Infectious Disease”, Second Edition, Eds., S. L., Gorbach, J. G., Bartlett, and N. R., Blacklow, W. B. Saunders Company, Philadelphia, (1998); which is hereby incorporated herein by reference).

Pheromones

In another embodiment, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability to synthesize and/or release a pheromone. Such a pheromone may, for example, alter the organisms behavior and/or metabolism.

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may modulate the biosynthesis and/or release of pheromones, the organisms ability to respond to pheromones (e.g., behaviorally, and/or metabolically), and/or the organisms ability to detect pheromones. Preferably, any of the pheromones, and/or volatiles released from the organism, or induced, by a polynucleotide or polypeptide and/or agonist or antagonist of the invention have behavioral effects the organism.

Other Activities

The polypeptide of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. These polypeptide may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulate neuronal growth and to treat, prevent, and/or diagnose neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. The polypeptide of the invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.

The polypeptide of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.

The polypeptide of the invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth. Along the same lines, the polypeptides of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.

The polypeptide of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues.

The polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.

The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.

The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, polypeptides or polynucleotides and/or agonist or antagonists of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive diseases, disorders, and/or conditions), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to increase the efficacy of a pharmaceutical composition, either directly or indirectly. Such a use may be administered in simultaneous conjunction with said pharmaceutical, or separately through either the same or different route of administration (e.g., intravenous for the polynucleotide or polypeptide of the present invention, and orally for the pharmaceutical, among others described herein.).

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to prepare individuals for extraterrestrial travel, low gravity environments, prolonged exposure to extraterrestrial radiation levels, low oxygen levels, reduction of metabolic activity, exposure to extraterrestrial pathogens, etc. Such a use may be administered either prior to an extraterrestrial event, during an extraterrestrial event, or both. Moreover, such a use may result in a number of beneficial changes in the recipient, such as, for example, any one of the following, non-limiting, effects: an increased level of hematopoietic cells, particularly red blood cells which would aid the recipient in coping with low oxygen levels; an increased level of B-cells, T-cells, antigen presenting cells, and/or macrophages, which would aid the recipient in coping with exposure to extraterrestrial pathogens, for example; a temporary (i.e., reversible) inhibition of hematopoietic cell production which would aid the recipient in coping with exposure to extraterrestrial radiation levels; increase and/or stability of bone mass which would aid the recipient in coping with low gravity environments; and/or decreased metabolism which would effectively facilitate the recipients ability to prolong their extraterrestrial travel by any one of the following, non-limiting means: (i) aid the recipient by decreasing their basal daily energy requirements; (ii) effectively lower the level of oxidative and/or metabolic stress in recipient (i.e., to enable recipient to cope with increased extraterrestial radiation levels by decreasing the level of internal oxidative/metabolic damage acquired during normal basal energy requirements; and/or (iii) enabling recipient to subsist at a lower metabolic temperature (i.e., cryogenic, and/or sub-cryogenic environment).

Also preferred is a method of treatment of an individual in need of an increased level of a protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

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EXAMPLES Description of the Preferred Embodiments Example 1 Bioinformatics Analysis

To search for novel proteases, a Hidden-Markov Model (HMM) of metalloproteases, Peptidase_M10 (obtained from the Pfam database in Sanger center) was used to search against the human genomic sequence database using the GENEWISEDB computer program (Genome Res. 10:547–8 (2000)). Genomic sequences that had a GENEWISEDB matching score of more than 15 against Peptidase_M10 were selected for further analysis. The genomic sequence contained in BAC (bacteria artificial chromosome) AL158835, AL360176 and AC023065 were found to contain putative exon sequences that were similar to metalloproteases. The portion of sequence from AL158835, AL360176 and AC023065 that matched Peptidase_M10 HMM profile were extracted and back-searched against non-redundant protein database using BLASTX program (Altschul et. al., 1990). The most similar protein sequence (Xenopus metalloprotease XMMP; Genbank Accession No. gilAAC21447; SEQ ID NO:3) was used as a template to predict more exons from AL158835, AL360176 and AC023065 using GENEWISEDB program (Birney and Durbin, 2000). The final predicted exons were assembled and a full-length clone of gene MMP-29 was obtained using the predicted exon sequences. The final predicted exons were assembled and a full length clone of gene MMP-29 was obtained using the predicted exon sequences.

The complete protein sequence of MMP-29 was found to have significant sequence homology with a family of known metalloproteinases. MMP-29 contains the sequence . . . HEIGHVLG LPH . . . (SEQ ID NO:88), fitting the consensus sequence pattern of . . . HE[ILF]GHXXGLXH . . . (SEQ ID NO:81) for all metallopoteinases. MMP-29 also contains an octapeptide . . . PRCGVPDM . . . (SEQ ID NO:89) that fits the highly conserved octapeptide pattern of . . . PRC[GN]XP[DR][LIVSAPKQ] . . . (SEQ ID NO:82) that has been shown to be involved in autoinhibition of metalloproteases (Breathnach R. et al, 1988; Navre M. et al. 1991). Between the autoinhibitive octapeptide and the catalytic peptide there are three pair of RR that possibly serve as the putative cleavage site for activation by furin proteinases.

Proteins in the metalloprotease family are secreted or transmembrane proteins. MMP-29 contains a strong signal sequence at the NH₂ terminal, suggesting that MMP-29 is a secreted protein. FIG. 3 is a Kyte-Doolittle hydrophobicity plot showing strong hydrophobic region at the NH₂-terminus of MMP-29. The signal peptide prediction tool named SPScan from GCG package also demonstrates an obvious cleavage site between residues 24 and 25 (FIG. 4). In conclusion, based on sequence similarity and known metalloproteinase signature sequences, the novel MMP-29 is a novel human metalloproteinase.

Example 2 Method for Constructing a Size Fractionated Brain and Testis cDNA Library

Brain and testis poly A+RNA was purchased from Clontech and converted into double stranded cDNA using the SuperScript™ Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies) except that no radioisotope was incorporated in either of the cDNA synthesis steps and that the cDNA was fractionated by HPLC. This was accomplished on a TransGenomics HPLC system equipped with a size exclusion column (TosoHass) with dimensions of 7.8 mm×30 cm and a particle size of 10 um. Tris buffered saline was used as the mobile phase and the column was run at a flow rate of 0.5 mL/min.

The resulting chromatograms were analyzed to determine which fractions should be pooled to obtain the largest cDNA's; generally fractions that eluted in the range of 12 to 15 minutes were pooled. The cDNA was precipitated prior to ligation into the Sal I/Not I sites in the pSport vector supplied with the kit. Using a combination of PCR with primers to the ends of the vector and Sal I/Not I restriction enzyme digestion of mini-prep DNA, it was determined that the average insert size of the library was greater the 3.5 Kb. The overall complexity of the library was greater that 10⁷ independent clones. The library was amplified in semi-solid agar for 2 days at 30° C. An aliquot (200 microliters) of the amplified library was inoculated into a 200 ml culture for single-stranded DNA isolation by super-infection with a f1 helper phage. After overnight growth, the released phage particles with precipitated with PEG and the DNA isolated with proteinase K, SDS and phenol extractions. The single stranded circular DNA was concentrated by ethanol precipitation and used for the cDNA capture experiments.

Example 3 Cloning of the Novel Human MMP-29 Metalloproteinase

Using the predict exon genomic sequence from bac AL158835, AL360176 and AC023065, an antisense 80 bp oligonucleotide with biotin on the 5′ end may be designed with the following sequence.

One microliter (one hundred and fifty nanograms) of the biotinylated oligonucleotide may be added to six microliters (six micrograms) of a mixture of single-stranded covalently closed circular brain and testis cDNA libraries and seven microliters of 100% formamide in a 0.5 ml PCR tube. The library, a mixture of the brain and testis cDNA library referenced in Example 2, in addition to, commercially available brain and testis cDNA libraries from Life Technologies, Rockville, Md. The mixture could then be heated in a thermal cycler to 95° C. for 2 mins. Fourteen microliters of 2× hybridization buffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2% SDS) was added to the heated probe/cDNA library mixture and incubated at 42° C. for 26 hours. Hybrids between the biotinylated oligonucleotide and the circular cDNA could be isolated by diluting the hybridization mixture to 220 microliters in a solution containing 1 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125 microliters of streptavidin magnetic beads. This solution would be incubated at 42° C. for 60 mins, mixing every 5 mins to resuspend the beads. The beads could be separated from the solution with a magnet and the beads washed three times in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs could be released from the biotinlyated oligonucleotide/streptavidin magnetic bead complex by adding 50 microliters of 0.1 N NaOH and incubating at room temperature for 10 mins. Six microliters of 3 M Sodium Acetate could be added along with 15 micrograms of glycogen and the solution ethanol precipitated with 120 microliters of 100% ethanol. The DNA would then be resuspended in 12 microliters of TE (10 mM Tris-HCl, pH 8.0), 1 mM EDTA, pH 8.0). The single stranded cDNA would be converted into double strands in a thermal cycler by mixing 5 microliters of the captured DNA with 1.5 microliters 10 micromolar standard SP6 primer (homologous to a sequence on the cDNA cloning vector) and 1.5 microliters of 10×PCR buffer. The mixture is then heated to 95° C. for 20 seconds then ramped down to 59° C. At this time 15 microliters of a repair mix, that is preheated to 70° C. (Repair mix contains 4 microliters of 5 mM dNTPs (1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25 microliters of water, and 0.25 microliters of Taq polymerase) is added. The solution is ramped back to 73° C. and incubated for 23 mins. The repaired DNA is ethanol precipitated and resuspended in 10 microliters of TE. Two microliters are then electroporated in E. coli DH12S cells and resulting colonies screened by PCR, using a primer pair designed from the genomic exonic sequence to identify the proper cDNAs.

Those cDNA clones that are positive by PCR would have their inserts sized and two clones would be chosen for DNA sequencing.

The full-length nucleotide sequence and the encoded polypeptide for MMP-29 is shown in FIGS. 1A–B.

Example 4 Expression Profiling of the Novel Human MMP-29 Metalloproteinase

The same PCR primer pair that was used to identify the novel MMP-29 cDNA clones (SEQ ID NO:30 and 31) was used to measure the steady state levels of mRNA by quantitative PCR. Briefly, first strand cDNA was made from commercially available mRNA. The relative amount of cDNA used in each assay was determined by performing a parallel experiment using a primer pair for a gene expressed in equal amounts in all tissues, cyclophilin. The cyclophilin primer pair detected small variations in the amount of cDNA in each sample and these data were used for normalization of the data obtained with the primer pair for the novel MMP-29. The PCR data was converted into a relative assessment of the difference in transcript abundance amongst the tissues tested and the data is presented in FIG. 5. Transcripts corresponding to MMP-29 were expressed highly in the spinal cord, and to a lesser extent in liver, thymus, brain, kidney, spleen, lung, small intestine, and bone marrow.

Example 5 Method of Assessing the Expression Profile of the Novel MMP-29 Polypeptides of the Present Invention Using Expanded mRNA Tissue and Cell Sources

Total RNA from tissues was isolated using the TriZol protocol (Invitrogen) and quantified by determining its absorbance at 260 nM. An assessment of the 18s and 28s ribosomal RNA bands was made by denaturing gel electrophoresis to determine RNA integrity.

The specific sequence to be measured was aligned with related genes found in GenBank to identity regions of significant sequence divergence to maximize primer and probe specificity. Gene-specific primers and probes were designed using the ABI primer express software to amplify small amplicons (150 base pairs or less) to maximize the likelihood that the primers function at 100% efficiency. All primer/probe sequences were searched against Public Genbank databases to ensure target specificity. Primers and probes were obtained from ABI.

For MMP-29, the primer probe sequences were as follows

Forward Primer 5′-GGGAGCGGGCAGGAGTT-3′ (SEQ ID NO:68) Reverse Primer 5′-TGCTCGTCGTCGTCAAAGTG-3′ (SEQ ID NO:69) TaqMan Probe 5′-CACACGCCTGGCGCCTAGGTG-3′ (SEQ ID NO:70) DNA Contamination

To access the level of contaminating genomic DNA in the RNA, the RNA was divided into 2 aliquots and one half was treated with Rnase-free Dnase (Invitrogen). Samples from both the Dnase-treated and non-treated were then subjected to reverse transcription reactions with (RT+) and without (RT−) the presence of reverse transcriptase. TaqMan assays were carried out with gene-specific primers (see above) and the contribution of genomic DNA to the signal detected was evaluated by comparing the threshold cycles obtained with the RT+/RT− non-Dnase treated RNA to that on the RT+/RT− Dnase treated RNA. The amount of signal contributed by genomic DNA in the Dnased RT− RNA must be less that 10% of that obtained with Dnased RT+ RNA. If not the RNA was not used in actual experiments.

Reverse Transcription Reaction and Sequence Detection

100 ng of Dnase-treated total RNA was annealed to 2.5 μM of the respective gene-specific reverse primer in the presence of 5.5 mM Magnesium Chloride by heating the sample to 72° C. for 2 min and then cooling to 55° C. for 30 min. 1.25 U/μl of MuLv reverse transcriptase and 500 μM of each dNTP was added to the reaction and the tube was incubated at 37° C. for 30 min. The sample was then heated to 90° C. for 5 min to denature enzyme.

Quantitative sequence detection was carried out on an ABI PRISM 7700 by adding to the reverse transcribed reaction 2.5 μM forward and reverse primers, 500 μM of each dNTP, buffer and 5U AmpliTaq Gold™. The PCR reaction was then held at 94° C. for 12 min, followed by 40 cycles of 94° C. for 15 sec and 60° C. for 30 sec.

Data Handling

The threshold cycle (Ct) of the lowest expressing tissue (the highest Ct value) was used as the baseline of expression and all other tissues were expressed as the relative abundance to that tissue by calculating the difference in Ct value between the baseline and the other tissues and using it as the exponent in 2^((ΔCt))

The expanded expression profile of the MMP-29 polypeptide is provided in FIG. 6 and described elsewhere herein.

Example 6 Method of Measuring the Protease Activity of MMP-29 Polypeptides.

Protease activity of the MMP-29 polypeptide may be measured by following the inhibition of proteolytic activity in cells, tissues, and/or in in vitro assays. In vitro assays for measuring protease activity using synthetic peptide fluorescent, spectrophotometric either through the use of single substrates (see below for examples), and fluorescence resonance transfer assays are well described in the art, as single substrates or as part of substrate libraries (Backes et al., 2000; Knight, C. G. Fluorimetric Assays of Proteolytic Enzymes. Meth. Enzymol. 248: 18–34 (1995)). In addition the proteolytic activity could be measured by following production of peptide products. Such approaches are well known to those familiar with the art (reviewed in McGeehan, G. M., Bickett, D. M., Wiseman, J. S., Green, M., Berman, Meth. Enzymol. 248: 35–46 (1995))

Inhibitor Identification

The MMP-29 may be incubated with potential inhibitors (preferably small molecule inhibitors or antibodies provided elsewhere herein) for different times and with varying concentrations. Residual protease activity could then be measured according to any appropriate means known in the art. Enzyme activity in the presence of control may be expressed as fraction of control and curve fit to pre-incubation time and protease concentration to determine inhibitory parameters including concentration that half maximally inhibits the enzyme activity.

Non-limiting examples of protease assays are well described in the art (Balasubramanian et al., 1993; Combrink et al., 1998). An example of a spectrophotometric protease assay is the Factor Xa assay. Briefly, human FXa (Calbiochem #233526) enzymatic activity is measured in a buffer containing 0.145 M NaCl, 0.005 M KCl, 1 mg/ml Polyethylene Glycol (PEG-8000), 0.030 M HEPES (pH 7.4) using 96-well microtiter plates (Nunc Immuno #439454). The enzyme is incubated with the protease at room temperature for varying amounts of time prior to starting the reaction with 100 μM S-2222 (phenyl-Ile-Glu-Gly-Arg-pNA, K_(m)=137 μM). The K_(m) for this, and other substrates, may be determined experimentally by measuring the enzyme activity at different substrate concentrations and curve fitting the data using Kaleidagraph V. Time-dependent optical density change may be followed at 405 nm using a kinetic microplate reader (Molecular Devices UVmax) at room temperature.

An example of a fluorescence assay which may be used for the present invention is the Factor VIIa assay. Briefly, the Factor VIIa assay is measured in the presence of human recombinant tissue factor (INNOVIN from Dade Behring Cat.# B4212–100). Human Factor Vlla may be obtained from Enzyme Research Labs (Cat.# HFVIIA 1640). Enzymatic activity could be measured in a buffer containing 150 nM NaCl, 5 mM CaCl₂, 1 mM CHAPS and 1 mg/ml PEG 6000 (pH 7.4) with 1 nM FVIIa and 100 μM D-IIe-Pro-Arg-AFC (Enzyme Systems Products, Km>200 μM) 0.66% DMSO. The assay (302 μl total volume) may be incubated at room temperature for 2 hr prior to reading fluorometric signal (Ex 405/Em 535) using a Victor 2 (Wallac) fluorescent plate reader.

In addition to the methods described above, protease activity (and therefore metalloproteinase activity) can be measured using fluorescent resonance energy transer (FRET with Quencher -P_(n)-P₃-P₂-P₁- -P₁′-P₂′- Fluorophore), fluorescent peptide bound to beads (Fluorophore -P_(n)-P₃-P₂-P₁- -P₁′-P₂′-Bead), dye-protein substrates and protease gel shifts. All of which are well known to those skilled in the art (see a non-limiting review in Knight, C. G. Fluorimetric Assays of Proteolytic Enzymes. Meth. Enzymol. 248: 18–34 (1995))

Additional assays, in addition to, assay methods are known in the art and are encompassed by the present invention. See, for example, Backes B J, Harris J L, Leonetti F, Craik C S, Ellman J A. Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. Nat Biotechnol. 18:187–93 (2000); Balasubramanian, N., St. Laurent, D. R., Federici, M. E., Meanwell, N. A., Wright, J. J., Schumacher, W. A., and Seiler, S. M. Active site-directed synthetic thrombin inhibitors: synthesis, in vitro and in vivo activity profile of BMY 44621 and analogs. an examination of the role of the amino group in the D-Phe-Pro-Arg-H series. J. Med. Chem. 36:300–303 (1993); and Combrink, K. D., Guilgeze, H. B., Meanwell, N. A., Pearce, B. C. Zulan, P., Bisacchi, G. S., Roberts, D. G. M., Stanley, P. Seiler, S. M. Novel 1,2-Benzisothiazol-3-one-1,1-dioxide Inhibitors of Human Mast Cell Tryptase. J. Med. Chem. 41:4854–4860 (1998); which are hereby incorporated herein by reference in their entirety.

Example 7 Determination of the Preferred Substrate Sequence of the MMP-29 Protease.

The preferred substrate sequence specificity of the MMP-29 metalloprotease of the present invention may be determined using using two redundant peptide libraries and Edman peptide sequencing (1–2) as shown below.

Protease Substrate Consensus Sequence Determination

The first peptide library is random, can vary in length and incorporates a modification at the N-terminus to block Edman sequencing. In the example provided, biotin is used as the blocking group. Proteolytic cleavage of the library is allowed to proceed long enough to turn over approximately 5–10% of the library. Edman sequencing of the peptide mixture provides the preferred substrate residues for the P′ sites on the protease. The second peptide library has fixed P′ residues to restrict the proteolytic cleavage site and an affinity tag for removing the C-terminal product of the proteolysis, leaving the N-terminal peptide product pool behind for Edman sequencing to determine the amino acid residues preferred in the P1, P2, P3 etc . . . sites of the protease.

Reagents.

The endoproteases Factor Xa (New England BioLabs, Inc., Beverly, Mass.) and human kidney Renin (Calbiochem, San Diego, Calif.) were purchased for validation experiments. A hexapeptide library containing 4.7×10⁷ peptide species was synthesized by the Molecular Redesign group (Natarajan & Riexinger) at Bristol-Myers Squibb Company (Princeton, N.J.). The library contained equivalent representation of 19 amino acid residues at each of the six degenerate positions and incorporated an N-terminal biotin group and a C-terminal amide. Cysteine residues were excluded from the peptide pool and Methionine residues were replaced with Norleucine.

Endoprotease Cleavage of the Peptide Library.

The following method may be used to determine the preferred substrate sequence downstream of the cleavage site. A 1.88 mM peptide library solution is prepared in phosphate buffer (10 mM Sodium Phosphate (pH 7.6), 0.1 M NaCl, and 10% DMSO) and is incubated with 2–30 μg endoprotease at 37° C. Using a fluorescamine assay to estimate the extent of peptide cleavage, the reaction is stopped at 5–10% completion with incubation at 100° C. for 2.0 minutes. Peptide pools are subjected to Edman sequencing. The data obtained is normalized and corrected for differences in efficiency of cleavage and recovery in the sequencer.

Fluorescamine Assay to Monitor Peptide Cleavage.

Primary amines generated during peptide cleavage is measured by reaction with fluorescamine (Aldrich, St. Louis, Mo.), as described in reference 3. The relative fluorescence is determined by measuring signals at □^(ex)=355 nm and □^(em)=460 nm on a PerkinElmer Wallac 1420 Spectrofluorometer. Reactions are sampled at multiple time points and assayed in triplicate. The amount of cleavage product formed is determined using the relative fluorescence produced by varying concentrations of a peptide standard of known concentration.

References.

-   (1) “Substrate Specificity of Cathepsins D and E Determined by     N-Terminal and C-Terminal Sequencing of Peptide Pools” D. Arnold et     al. (1997) Eur. J. Biochem. 249, 171. -   (2) “Determination of Protease Cleavage Site Motifs Using     Mixture-Based Oriented Peptide Libraries” B. E. Turk et al. (2001)     Nature Biotech. 19, 661. -   (3) “Fluorescamine: a Reagent for Assay of Amino Acids, Peptides,     Proteins, and Primary Amines in the Picomole Range” S.     Udenfirend, S. Stein, P. Bohlen, W.

Dairman, W. Leimgruber, and M. Weigele (1972) Science 178, 87.

Example 8 Method of Screening for Compounds That Interact With the MMP-29 Polypeptide

The following assays are designed to identify compounds that bind to the MMP-29 polypeptide, bind to other cellular proteins that interact with the MMP-29 polypeptide, and to compounds that interfere with the interaction of the MMP-29 polypeptide with other cellular proteins.

Such compounds can include, but are not limited to, other cellular proteins. Specifically, such compounds can include, but are not limited to, peptides, such as, for example, soluble peptides, including, but not limited to Ig-tailed fusion peptides, comprising extracellular portions of MMP-29 polypeptide transmembrane receptors, and members of random peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature 354:82–84; Houghton, R. et al., 1991, Nature 354:84–86), made of D- and/or L-configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate phosphopeptide libraries; see, e.g., Songyang, Z., et al., 1993, Cell 72:767–778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′).sub.2 and FAb expression libary fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.

Compounds identified via assays such as those described herein can be useful, for example, in elaborating the biological function of the MMP-29 polypeptide, and for ameliorating symptoms of tumor progression, for example. In instances, for example, whereby a tumor progression state or disorder results from a lower overall level of MMP-29 expression, MMP-29 polypeptide and/or MMP-29 polypeptide activity in a cell involved in the tumor progression state or disorder, compounds that interact with the MMP-29 polypeptide can include ones which accentuate or amplify the activity of the bound MMP-29 polypeptide. Such compounds would bring about an effective increase in the level of MMP-29 polypeptide activity, thus ameliorating symptoms of the tumor progression disorder or state. In instances whereby mutations within the MMP-29 polypeptide cause aberrant MMP-29 polypeptides to be made which have a deleterious effect that leads to tumor progression, compounds that bind MMP-29 polypeptide can be identified that inhibit the activity of the bound MMP-29 polypeptide. Assays for testing the effectiveness of such compounds are known in the art and discussed, elsewhere herein.

Example 9 Method of Screening, In Vitro, Compounds that Bind to the MMP-29 Polypeptide

In vitro systems can be designed to identify compounds capable of binding the MMP-29 polypeptide of the invention. Compounds identified can be useful, for example, in modulating the activity of wild type and/or mutant MMP-29 polypeptide, preferably mutant MMP-29 polypeptide, can be useful in elaborating the biological function of the MMP-29 polypeptide, can be utilized in screens for identifying compounds that disrupt normal MMP-29 polypeptide interactions, or can in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to the MMP-29 polypeptide involves preparing a reaction mixture of the MMP-29 polypeptide and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring MMP-29 polypeptide or the test substance onto a solid phase and detecting MMP-29 polypeptide/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the MMP-29 polypeptide can be anchored onto a solid surface, and the test compound, which is not anchored, can he labeled, either directly or indirectly.

In practice, microtitre plates can conveniently be utilized as the solid phase. The anchored component can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid surface. The surfaces can be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for MMP-29 polypeptide or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.

Example 10 Method of Identifying Compounds that Interfere with MMP-29 Polypeptide/Cellular Product Interaction

The MMP-29 polypeptide of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. Such macromolecules include, but are not limited to, nucleic acid molecules and those products identified via methods such as those described, elsewhere herein. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partner(s)”. For the purpose of the present invention, “binding partner” may also encompass small molecule compounds, polysaccarides, lipids, and any other molecule or molecule type referenced herein. Compounds that disrupt such interactions can be useful in regulating the activity of the MMP-29 polypeptide, especially mutant MMP-29 polypeptide. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and the like described in elsewhere herein.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the MMP-29 polypeptide and its cellular or extracellular binding partner or partners involves preparing a reaction mixture containing the MMP-29 polypeptide, and the binding partner under conditions and for a time sufficient to allow the two products to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of MMP-29 polypeptide and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the MMP-29 polypeptide and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the MMP-29 polypeptide and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal MMP-29 polypeptide can also be compared to complex formation within reaction mixtures containing the test compound and mutant MMP-29 polypeptide. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal MMP-29 polypeptide.

The assay for compounds that interfere with the interaction of the MMP-29 polypeptide and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the MMP-29 polypeptide or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the MMP-29 polypeptide and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the MMP-29 polypeptide and interactive cellular or extracellular binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

In a heterogeneous assay system, either the MMP-29 polypeptide or the interactive cellular or extracellular binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtitre plates are conveniently utilized. The anchored species can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished simply by coating the solid surface with a solution of the MMP-29 polypeptide or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface. The surfaces can be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the MMP-29 polypeptide and the interactive cellular or extracellular binding partner product is prepared in which either the MMP-29 polypeptide or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt MMP-29 polypeptide-cellular or extracellular binding partner interaction can be identified.

In a particular embodiment, the MMP-29 polypeptide can be prepared for immobilization using recombinant DNA techniques known in the art. For example, the MMP-29 polypeptide coding region can be fused to a glutathione-S-transferase (GST) gene using a fusion vector such as pGEX-5X-1, in such a manner that its binding activity is maintained in the resulting fusion product. The interactive cellular or extracellular product can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above. This antibody can be labeled with the radioactive isotope sup.125 I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-MMP-29 polypeptide fusion product can be anchored to glutathione-agarose beads. The interactive cellular or extracellular binding partner product can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between the MMP-29 polypeptide and the interactive cellular or extracellular binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-MMP-29 polypeptide fusion product and the interactive cellular or extracellular binding partner product can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the binding partners are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the MMP-29 polypeptide product and the interactive cellular or extracellular binding partner (in case where the binding partner is a product), in place of one or both of the full length products.

Any number of methods routinely practiced in the art can be used to identify and isolate the protein's binding site. These methods include, but are not limited to, mutagenesis of one of the genes encoding one of the products and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can be selected. Sequence analysis of the genes encoding the respective products will reveal the mutations that correspond to the region of the product involved in interactive binding. Alternatively, one product can be anchored to a solid surface using methods described in this Section above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain can remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the cellular or extracellular binding partner product is obtained, short gene segments can be engineered to express peptide fragments of the product, which can then be tested for binding activity and purified or synthesized.

Example 11 Isolation of a Specific Clone from the Deposited Sample

The deposited material in the sample assigned the ATCC Deposit Number cited in Table I for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone. Thus, deposits sharing the same ATCC Deposit Number contain at least a plasmid for each cDNA clone identified in Table I. Typically, each ATCC deposit sample cited in Table I comprises a mixture of approximately equal amounts (by weight) of about 1–10 plasmid DNAs, each containing a different cDNA clone and/or partial cDNA clone; but such a deposit sample may include plasmids for more or less than 2 cDNA clones.

Two approaches can be used to isolate a particular clone from the deposited sample of plasmid DNA(s) cited for that clone in Table I. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to SEQ ID NO:1.

Particularly, a specific polynucleotide with 30–40 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide is labeled, for instance, with 32P-(-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.

Alternatively, two primers of 17–20 nucleotides derived from both ends of the SEQ ID NO:1 (i.e., within the region of SEQ ID NO:1 bounded by the 5′ NT and the 3′ NT of the clone defined in Table I) are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 ul of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5–5 mM MgCl2, 0.01% (w/v) gelatin, 20 M each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.

The polynucleotide(s) of the present invention, the polynucleotide encoding the polypeptide of the present invention, or the polypeptide encoded by the deposited clone may represent partial. or incomplete versions of the complete coding region (i.e., full-length gene). Several methods are known in the art for the identification of the 5′ or 3′ non-coding and/or coding portions of a gene which may not be present in the deposited clone. The methods that follow are exemplary and should not be construed as limiting the scope of the invention. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5′ and 3′ “RACE” protocols that are well known in the art. For instance, a method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683–1684 (1993)).

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5′ portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full-length gene.

This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA that may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the desired gene. Moreover, it may be advantageous to optimize the RACE protocol to increase the probability of isolating additional 5′ or 3′ coding or non-coding sequences. Various methods of optimizing a RACE protocol are known in the art, though a detailed description summarizing these methods can be found in B. C. Schaefer, Anal. Biochem., 227:255–273, (1995).

An alternative method for carrying out 5′ or 3′ RACE for the identification of coding or non-coding sequences is provided by Frohman, M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998–9002 (1988). Briefly, a cDNA clone missing either the 5′ or 3′ end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start of translation, therefor. The following briefly describes a modification of this original 5′ RACE procedure. Poly A+ or total RNAs reverse transcribed with Superscript II (Gibco/BRL) and an antisense or I complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoIJ Sail and ClaI) at the 5′ end and a primer containing just these restriction sites. This double-stranded cDNA is PCR amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with XhoI or SalI, and ligated to a plasmid such as pBluescript SKII (Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the correct protein-coding inserts. Correct 5′ ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial kits are used to amplify and recover 3′ ends.

Several quality-controlled kits are commercially available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of full length genes. A second kit is available from Clontech which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227–32(1991). The major differences in procedure are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.

An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNA library double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.

RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to Obtain Full Length Genes

Once a gene of interest is identified, several methods are available for the identification of the 5′ or 3′ portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5′ and 3′RACE. While the full-length gene may be present in the library and can be identified by probing, a useful method for generating the 5′ or 3′ end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5′RACE is available for generating the missing 5′ end of a desired full-length gene. (This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7): 1683–1684 (1993)). Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably 30 containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest, is used to PCR amplify the 5′ portion of the desired full length gene which may then be sequenced and used to generate the full length gene. This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction can then be used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the apoptosis related of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the relevant apoptosis related.

Example 12 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 9, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, that expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. EPTG induces by inactivating the lacd repressor, clearing the P/O leading to increased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3–4 hours at 4 degree C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6× His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively. the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM imidazole. Imidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4 degree C. or frozen at −80 degree C.

Example 13 Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4–10 degree C.

Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4–10 degree C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.

The cells are then lysed by passing the solution through a microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at 4000–6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2–4 hours. After 7000×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4 degree C. overnight to allow further GuHCl extraction.

Following high speed centrifugation (30.000×g) to remove insoluble particles. the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM LDTA by vigorous stirring. The refolded diluted protein solution is kept at 4 degree C. without mixing for 12 hours prior to further purification steps.

To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 um membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perceptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perceptive Biosystems) and weak anion (Poros CM-20, Perceptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A280 monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Coomassie blue stained 16% SDS-PAGE gel when 5 ug of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.

Example 14 Cloning and Expression of a Polypeptide in a Baculovirus Expression System

In this example, the plasmid shuttle vector pAc373 is used to insert a polynucleotide into a baculovirus to express a polypeptide. A typical baculovirus expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites, which may include, for example BamHI, XbaI and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is often used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above, such as pVL941 and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31–39 (1989).

A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 9, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites at the 5′ end of the primers in order to clone the amplified product into the expression vector. Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified elsewhere herein (if applicable), is amplified using the PCR protocol described in Example 9. If the naturally occurring signal sequence is used to produce the protein, the vector used does not need a second signal peptide. Alternatively, the vector can be modified to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.

Five ug of a plasmid containing the polynucleotide is co-transformed with 1.0 ug of a commercially available linearized baculovirus DNA (“BaculoGoldtm baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413–7417 (1987). One ug of BaculoGoldtm virus DNA and 5 ug of the plasmid are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27 degrees C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27 degrees C. for four days.

After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9–10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ul of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of 35S-methionine and 5 uCi 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.

Example 15 Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transformation with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transformed cells.

The transformed gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. . . . 253:1357–1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107–143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64–68 (1991).) Another useful selection marker is the enzyme glutanine synthase (GS) (Murphy et al., Biochem J. 227:277–279 (1991); Bebbington et al., Bio/Technology 10:169–175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.

A polynucleotide of the present invention is amplified according to the protocol outlined in herein. If the naturally occurring signal sequence is used to produce the protein, the vector does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.) The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used for transformation. Five ug of an expression plasmid is cotransformed with 0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10–14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100–200 uM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.

Example 16 Protein Fusions

The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example described herein; see also EP A 394,827; Traunecker, et al., Nature 331:84–86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases the half-life time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule.

Briefly, the human Fe portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.

The naturally occurring signal sequence may be used to produce the protein (if applicable). Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat. No. 6,066,781, supra.)

Human IgG Fc region:

(SEQ ID NO:29)     GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACC GTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCC CAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGC GTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC AGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCT CCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG AACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGC CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGC CTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGAT GCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 17 Method of Creating N- and C-terminal Deletion Mutants Corresponding to the MMP-29 Polypeptide of the Present Invention

As described elsewhere herein, the present invention encompasses the creation of N- and C-terminal deletion mutants, in addition to any combination of N- and C-terminal deletions thereof, corresponding to the MMP-29 polypeptide of the present invention. A number of methods are available to one skilled in the art for creating such mutants. Such methods may include a combination of PCR amplification and gene cloning methodology. Although one of skill in the art of molecular biology, through the use of the teachings provided or referenced herein, and/or otherwise known in the art as standard methods, could readily create each deletion mutant of the present invention, exemplary methods are described below.

Briefly, using the isolated cDNA clone encoding the full-length MMP-29 polypeptide sequence (as described in Example 9, for example), appropriate primers of about 15–25 nucleotides derived from the desired 5′ and 3′ positions of SEQ ID NO:1 may be designed to PCR amplify, and subsequently clone, the intended N- and/or C-terminal deletion mutant. Such primers could comprise, for example, an inititation and stop codon for the 5′ and 3′ primer, respectively. Such primers may also comprise restriction sites to facilitate cloning of the deletion mutant post amplification. Moreover, the primers may comprise additional sequences, such as, for example, flag-tag sequences, kozac sequences, or other sequences discussed and/or referenced herein.

For example, in the case of the E25 to M569 N-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant:

5′Primer 5′-GCAGCA GCGGCCGC GAGAGTCTCTTCCACAGCCGGGACC-3′ (SEQ ID NO:29)             NotI 3′Primer 5′-GCAGCA GTCGAC CATGTTCAGTGTGGAGATATGGACG-3′ (SEQ ID NO:30)            SalI

For example, in the case of the M1 to N500 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant:

5′Primer 5′-GCAGCA GCGGCCGC ATGCTCGCCGCCTCCATCTTCCGTC-3′ (SEQ ID NO:31)             NotI 3′Primer 5′-GCAGCA GTCGAC ATTTTGTGGTATTACTGCTGG-3′ (SEQ ID NO:32)            SalI

Representative PCR amplification conditions are provided below, although the skilled artisan would appreciate that other conditions may be required for efficient amplification. A 100 ul PCR reaction mixture may be prepared using 10 ng of the template DNA (cDNA clone of MMP-29), 200 uM 4dNTPs, 1 uM primers, 0.25U Taq DNA polymerase (PE), and standard Taq DNA polymerase buffer. Typical PCR cycling condition are as follows:

20–25 cycles: 45 sec, 93 degrees  2 min, 50 degrees  2 min, 72 degrees 1 cycle: 10 min, 72 degrees

After the final extension step of PCR, 5U Klenow Fragment may be added and incubated for 15 min at 30 degrees.

Upon digestion of the fragment with the NotI and SalI restriction enzymes, the fragment could be cloned into an appropriate expression and/or cloning vector which has been similarly digested (e.g., pSport1, among others). The skilled artisan would appreciate that other plasmids could be equally substituted, and may be desirable in certain circumstances. The digested fragment and vector are then ligated using a DNA ligase, and then used to transform competent E. coli cells using methods provided herein and/or otherwise known in the art.

The 5′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the MMP-29 gene (SEQ ID NO:1), and ‘X’ is equal to the most N-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 5′ primer, while the second term will provide the end 3′ nucleotide position of the 5′ primer corresponding to sense strand of SEQ ID NO:1. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 5′ primer may be desired in certain circumstances (e.g., kozac sequences, etc.).

The 3′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))−25), wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the MMP-29 gene (SEQ ID NO:1), and ‘X’ is equal to the most C-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 3′ primer, while the second term will provide the end 3′ nucleotide position of the 3′ primer corresponding to the anti-sense strand of SEQ ID NO:1. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 3′ primer may be desired in certain circumstances (e.g., stop codon sequences, etc.). The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.

The same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any C-terminal deletion mutant of the present invention. Moreover, the same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any combination of N-terminal and C-terminal deletion mutant of the present invention. The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.

Example 18 Regulation of Protein Expression Via Controlled Aggregation in the Endoplasmic Reticulum

As described more particularly herein, proteins regulate diverse cellular processes in higher organisms, ranging from rapid metabolic changes to growth and differentiation. Increased production of specific proteins could be used to prevent certain diseases and/or disease states. Thus, the ability to modulate the expression of specific proteins in an organism would provide significant benefits.

Numerous methods have been developed to date for introducing foreign genes, either under the control of an inducible, constitutively active, or endogenous promoter, into organisms. of particular interest are the inducible promoters (see, M. Gossen, et al., Proc. Natl. Acad. Sci. USA., 89:5547 (1992); Y. Wang, et al., Proc. Natl. Acad. Sci. USA, 91:8180 (1994), D. No., et al., Proc. Natl. Acad. Sci. USA, 93:3346 (1996); and V. M. Rivera, et al., Nature Med, 2:1028 (1996); in addition to additional examples disclosed elsewhere herein). In one example, the gene for erthropoietin (Epo) was transferred into mice and primates under the control of a small molecule inducer for expression (e.g., tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512, (1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M. Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X. Ye et al., Science, 283:88 (1999). Although such systems enable efficient induction of the gene of interest in the organism upon addition of the inducing agent (i.e., tetracycline, rapamycin, etc,.), the levels of expression tend to peak at 24 hours and trail off to background levels after 4 to 14 days. Thus, controlled transient expression is virtually impossible using these systems, though such control would be desirable.

A new alternative method of controlling gene expression levels of a protein from a transgene (i.e., includes stable and transient transformants) has recently been elucidated (V. M. Rivera., et al., Science, 287:826–830, (2000)). This method does not control gene expression at the level of the mRNA like the aforementioned systems. Rather, the system controls the level of protein in an active secreted form. In the absence of the inducing agent, the protein aggregates in the ER and is not secreted. However, addition of the inducing agent results in dis-aggregation of the protein and the subsequent secretion from the ER. Such a system affords low basal secretion, rapid, high level secretion in the presence of the inducing agent, and rapid cessation of secretion upon removal of the inducing agent. In fact, protein secretion reached a maximum level within 30 minutes of induction, and a rapid cessation of secretion within 1 hour of removing the inducing agent. The method is also applicable for controlling the level of production for membrane proteins.

Detailed methods are presented in V. M. Rivera., et al., Science, 287:826–830, (2000)), briefly:

Fusion protein constructs are created using polynucleotide sequences of the present invention with one or more copies (preferably at least 2, 3, 4, or more) of a conditional aggregation domain (CAD) a domain that interacts with itself in a ligand-reversible manner (i.e., in the presence of an inducing agent) using molecular biology methods known in the art and discussed elsewhere herein. The CAD domain may be the mutant domain isolated from the human FKBP12 (Phe³⁶ to Met) protein (as disclosed in V. M. Rivera., et al., Science, 287:826–830, (2000), or alternatively other proteins having domains with similar ligand-reversible, self-aggregation properties. As a principle of design the fusion protein vector would contain a furin cleavage sequence operably linked between the polynucleotides of the present invention and the CAD domains. Such a cleavage site would enable the proteolytic cleavage of the CAD domains from the polypeptide of the present invention subsequent to secretion from the ER and upon entry into the trans-Golgi (J. B. Denault, et al., FEBS Lett., 379:113, (1996)). Alternatively, the skilled artisan would recognize that any proteolytic cleavage sequence could be substituted for the furin sequence provided the substituted sequence is cleavable either endogenously (e.g., the furin sequence) or exogenously (e.g., post secretion, post purification, post production, etc.). The preferred sequence of each feature of the fusion protein construct, from the 5′ to 3′ direction with each feature being operably linked to the other, would be a promoter, signal sequence, “X” number of (CAD)x domains, the furin sequence (or other proteolytic sequence), and the coding sequence of the polypeptide of the present invention. The artisan would appreciate that the promotor and signal sequence, independent from the other, could be either the endogenous promotor or signal sequence of a polypeptide of the present invention, or alternatively, could be a heterologous signal sequence and promotor.

The specific methods described herein for controlling protein secretion levels through controlled ER aggregation are not meant to be limiting are would be generally applicable to any of the polynucleotides and polypeptides of the present invention, including variants, homologues, orthologs, and fragments therein.

Example 19 Alteration of Protein Glycosylation Sites to Enhance Characteristics of Polypeptides of the Invention

Many eukaryotic cell surface and proteins are post-translationally processed to incorporate N-linked and O-linked carbohydrates (Komfeld and Kornfeld (1985) Annu. Rev. Biochem. 54:631–64; Rademacher et al., (1988) Annu. Rev. Biochem. 57:785–838). Protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion (Fieldler and Simons (1995) Cell, 81:309–312; Helenius (1994) Mol. Biol. of the Cell 5:253–265; Olden et al., (1978) Cell, 13:461–473; Caton et al., (1982) Cell, 37:417–427; Alexander and Elder (1984), Science, 226:1328–1330; and Flack et al., (1994), J. Biol. Chem., 269:14015–14020). In higher organisms, the nature and extent of glycosylation can markedly affect the circulating half-life and bio-availability of proteins by mechanisms involving receptor mediated uptake and clearance (Ashwell and Morrell, (1974), Adv. Enzymol., 41:99–128; Ashwell and Harford (1982), Ann. Rev. Biochem., 51:531–54). Receptor systems have been identified that are thought to play a major role in the clearance of serum proteins through recognition of various carbohydrate structures on the glycoproteins (Stockert (1995), Physiol. Rev., 75:591–609; Kery et al., (1992), Arch. Biochem. Biophys., 298:49–55). Thus, production strategies resulting in incomplete attachment of terminal sialic acid residues might provide a means of shortening the bioavailability and half-life of glycoproteins. Conversely, expression strategies resulting in saturation of terminal sialic acid attachment sites might lengthen protein bioavailability and half-life.

In the development of recombinant glycoproteins for use as pharmaceutical products, for example, it has been speculated that the pharmacodynamics of recombinant proteins can be modulated by the addition or deletion of glycosylation sites from a glycoproteins primary structure (Berman and Lasky (1985a) Trends in Biotechnol., 3:51–53). However, studies have reported that the deletion of N-linked glycosylation sites often impairs intracellular transport and results in the intracellular accumulation of glycosylation site variants (Machamer and Rose (1988), J. Biol Chem., 263:5955–5960; Gallagher et al., (1992), J. Virology., 66:7136–7145; Collier et al., (1993), Biochem., 32:7818–7823; Claffey et al., (1995) Biochemica et Biophysica Acta, 1246:1–9; Dube et al., (1988), J. Biol. Chem. 263:17516–17521). While glycosylation site variants of proteins can be expressed intracellularly, it has proved difficult to recover useful quantities from growth conditioned cell culture medium.

Moreover, it is unclear to what extent a glycosylation site in one species will be recognized by another species glycosylation machinery. Due to the importance of glycosylation in protein metabolism, particularly the secretion and/or expression of the protein, whether a glycosylation signal is recognized may profoundly determine a proteins ability to be expressed, either endogenously or recombinately, in another organism (i.e., expressing a human protein in E. coli, yeast, or viral organisms; or an E. coli, yeast, or viral protein in human, etc.). Thus, it may be desirable to add, delete, or modify a glycosylation site, and possibly add a glycosylation site of one species to a protein of another species to improve the proteins functional, bioprocess purification, and/or structural characteristics (e.g., a polypeptide of the present invention).

A number of methods may be employed to identify the location of glycosylation sites within a protein. One preferred method is to run the translated protein sequence through the PROSITE computer program (Swiss Institute of Bioinformatics). Once identified, the sites could be systematically deleted, or impaired, at the level of the DNA using mutagenesis methodology known in the art and available to the skilled artisan, Preferably using PCR-directed mutagenesis (See Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). Similarly, glycosylation sites could be added, or modified at the level of the DNA using similar methods, preferably PCR methods (See, Maniatis, supra). The results of modifying the glycosylation sites for a particular protein (e.g., solubility, secretion potential, activity, aggregation, proteolytic resistance, etc.) could then be analyzed using methods know in the art.

The skilled artisan would acknowledge the existence of other computer algorithms capable of predicting the location of glycosylation sites within a protein. For example, the Motif computer program (Genetics Computer Group suite of programs) provides this function, as well.

Example 20 Method of Enhancing the Biological Activity/Functional Characteristics of Invention through Molecular Evolution

Although many of the most biologically active proteins known are highly effective for their specified function in an organism, they often possess characteristics that make them undesirable for transgenic, therapeutic, and/or industrial applications. Among these traits, a short physiological half-life is the most prominent problem, and is present either at the level of the protein, or the level of the proteins mRNA. The ability to extend the half-life, for example, would be particularly important for a proteins use in gene therapy, transgenic animal production, the bioprocess production and purification of the protein, and use of the protein as a chemical modulator among others. Therefore, there is a need to identify novel variants of isolated proteins possessing characteristics which enhance their application as a therapeutic for treating diseases of animal origin, in addition to the proteins applicability to common industrial and pharmaceutical applications.

Thus, one aspect of the present invention relates to the ability to enhance specific characteristics of invention through directed molecular evolution. Such an enhancement may, in a non-limiting example, benefit the inventions utility as an essential component in a kit, the inventions physical attributes such as its solubility, structure, or codon optimization, the inventions specific biological activity, including any associated enzymatic activity, the proteins enzyme kinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity, protein-DNA binding activity, antagonist/inhibitory activity (including direct or indirect interaction), agonist activity (including direct or indirect interaction), the proteins antigenicity (e.g., where it would be desirable to either increase or decrease the antigenic potential of the protein), the immunogenicity of the protein, the ability of the protein to form dimers, trimers, or multimers with either itself or other proteins, the antigenic efficacy of the invention, including its subsequent use a preventative treatment for disease or disease states, or as an effector for targeting diseased genes. Moreover, the ability to enhance specific characteristics of a protein may also be applicable to changing the characterized activity of an enzyme to an activity completely unrelated to its initially characterized activity. Other desirable enhancements of the invention would be specific to each individual protein, and would thus be well known in the art and contemplated by the present invention.

For example, an engineered metalloproteinase may be constitutively active upon binding of its cognate ligand. Alternatively, an engineered metalloproteinase may be constitutively active in the absence of substrate binding. In yet another example, an engineered metalloproteinase may be capable of being activated with less than all of the regulatory factors and/or conditions typically required for metalloproteinase activation (e.g., substrate binding, presence of a zinc ion, phosphorylation, conformational changes, etc.). Such metalloproteinases would be useful in screens to identify metalloproteinase modulators, among other uses described herein.

Directed evolution is comprised of several steps. The first step is to establish a library of variants for the gene or protein of interest. The most important step is to then select for those variants that entail the activity you wish to identify. The design of the screen is essential since your screen should be selective enough to eliminate non-useful variants, but not so stringent as to eliminate all variants. The last step is then to repeat the above steps using the best variant from the previous screen. Each successive cycle, can then be tailored as necessary, such as increasing the stringency of the screen, for example.

Over the years, there have been a number of methods developed to introduce mutations into macromolecules. Some of these methods include, random mutagenesis, “error-prone” PCR, chemical mutagenesis, site-directed mutagenesis, and other methods well known in the art (for a comprehensive listing of current mutagenesis methods, see Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). Typically, such methods have been used, for example, as tools for identifying the core functional region(s) of a protein or the function of specific domains of a protein (if a multi-domain protein). However, such methods have more recently been applied to the identification of macromolecule variants with specific or enhanced characteristics.

Random mutagenesis has been the most widely recognized method to date. Typically, this has been carried out either through the use of “error-prone” PCR (as described in Moore, J., et al, Nature Biotechnology 14:458, (1996), or through the application of randomized synthetic oligonucleotides corresponding to specific regions of interest (as described by Derbyshire, K. M. et al, Gene, 46:145–152, (1986), and Hill, D E, et al, Methods Enzymol., 55:559–568, (1987). Both approaches have limits to the level of mutagenesis that can be obtained. However, either approach enables the investigator to effectively control the rate of mutagenesis. This is particularly important considering the fact that mutations beneficial to the activity of the enzyme are fairly rare. In fact, using too high a level of mutagenesis may counter or inhibit the desired benefit of a useful mutation.

While both of the aforementioned methods are effective for creating randomized pools of macromolecule variants, a third method, termed “DNA Shuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747, (1994)) has recently been elucidated. DNA shuffling has also been referred to as “directed molecular evolution”, “exon-shuffling”, “directed enzyme evolution”, “in vitro evolution”, and “artificial evolution”. Such reference terms are known in the art and are encompassed by the invention. This new, preferred, method apparently overcomes the limitations of the previous methods in that it not only propagates positive traits, but simultaneously eliminates negative traits in the resulting progeny.

DNA shuffling accomplishes this task by combining the principal of in vitro recombination, along with the method of “error-prone” PCR. In effect, you begin with a randomly digested pool of small fragments of your gene, created by Dnase I digestion, and then introduce said random fragments into an “error-prone” PCR assembly reaction. During the PCR reaction, the randomly sized DNA fragments not only hybridize to their cognate strand, but also may hybridize to other DNA fragments corresponding to different regions of the polynucleotide of interest—regions not typically accessible via hybridization of the entire polynucleotide. Moreover, since the PCR assembly reaction utilizes “error-prone” PCR reaction conditions, random mutations are introduced during the DNA synthesis step of the PCR reaction for all of the fragments—further diversifying the potential hybridization sites during the annealing step of the reaction.

A variety of reaction conditions could be utilized to carry-out the DNA shuffling reaction. However, specific reaction conditions for DNA shuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:

Prepare the DNA substrate to be subjected to the DNA shuffling reaction. Preparation may be in the form of simply purifying the DNA from contaminating cellular material, chemicals, buffers, oligonucleotide primers, deoxynucleotides, RNAs, etc., and may entail the use of DNA purification kits as those provided by Qiagen, Inc., or by the Promega, Corp., for example.

Once the DNA substrate has been purified, it would be subjected to Dnase I digestion. About 2–4 ug of the DNA substrate(s) would be digested with 0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCL, pH 7.4/1 mM MgCl2 for 10–20 min. at room temperature. The resulting fragments of 10–50 bp could then be purified by running them through a 2% low-melting point agarose gel by electrophoresis onto DE81 ion-exchange paper (Whatmann) or could be purified using Microcon concentrators (Amicon) of the appropriate molecular weight cutoff, or could use oligonucleotide purification columns (Qiagen), in addition to other methods known in the art. If using DE81 ion-exchange paper, the 10–50 bp fragments could be eluted from said paper using 1M NaCl, followed by ethanol precipitation.

The resulting purified fragments would then be subjected to a PCR assembly reaction by re-suspension in a PCR mixture containing: 2 mM of each dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris•HCL, pH 9.0, and 0.1% Triton X-100, at a final fragment concentration of 10–30 ng/ul. No primers are added at this point. Taq DNA polymerase (Promega) would be used at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 C for 60 s; 94 C for 30 s, 50–55 C for 30 s, and 72 C for 30s using 30–45 cycles, followed by 72 C for 5 min using an MJ Research (Cambridge, Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a 1:40 dilution of the resulting primeness product would then be introduced into a PCR mixture (using the same buffer mixture used for the assembly reaction) containing 0.8 um of each primer and subjecting this mixture to 15 cycles of PCR (using 94 C for 30 s, 50 C for 30 s, and 72 C for 30 s). The referred primers would be primers corresponding to the nucleic acid sequences of the polynucleotide(s) utilized in the shuffling reaction. Said primers could consist of modified nucleic acid base pairs using methods known in the art and referred to else where herein, or could contain additional sequences (i.e., for adding restriction sites, mutating specific base-pairs, etc.).

The resulting shuffled, assembled, and amplified product can be purified using methods well known in the art (e.g., Qiagen PCR purification kits) and then subsequently cloned using appropriate restriction enzymes.

Although a number of variations of DNA shuffling have been published to date, such variations would be obvious to the skilled artisan and are encompassed by the invention. The DNA shuffling method can also be tailored to the desired level of mutagenesis using the methods described by Zhao, et al. (Nucl Acid Res., 25(6):1307–1308, (1997).

As described above, once the randomized pool has been created, it can then be subjected to a specific screen to identify the variant possessing the desired characteristic(s). Once the variant has been identified, DNA corresponding to the variant could then be used as the DNA substrate for initiating another round of DNA shuffling. This cycle of shuffling, selecting the optimized variant of interest, and then re-shuffling, can be repeated until the ultimate variant is obtained. Examples of model screens applied to identify variants created using DNA shuffling technology may be found in the following publications: J. C., Moore, et al., J. Mol. Biol., 272:336–347, (1997), F. R., Cross, et al., Mol. Cell. Biol., 18:2923–2931, (1998), and A. Crameri., et al., Nat. Biotech., 15:436–438, (1997).

DNA shuffling has several advantages. First, it makes use of beneficial mutations. When combined with screening, DNA shuffling allows the discovery of the best mutational combinations and does not assume that the best combination contains all the mutations in a population. Secondly, recombination occurs simultaneously with point mutagenesis. An effect of forcing DNA polymerase to synthesize full-length genes from the small fragment DNA pool is a background mutagenesis rate. In combination with a stringent selection method, enzymatic activity has been evolved up to 16000 fold increase over the wild-type form of the enzyme. In essence, the background mutagenesis yielded the genetic variability on which recombination acted to enhance the activity.

A third feature of recombination is that it can be used to remove deleterious mutations. As discussed above, during the process of the randomization, for every one beneficial mutation, there may be at least one or more neutral or inhibitory mutations. Such mutations can be removed by including in the assembly reaction an excess of the wild-type random-size fragments, in addition to the random-size fragments of the selected mutant from the previous selection. During the next selection, some of the most active variants of the polynucleotide/polypeptide/enzyme, should have lost the inhibitory mutations.

Finally, recombination enables parallel processing. This represents a significant advantage since there are likely multiple characteristics that would make a protein more desirable (e.g. solubility, activity, etc.). Since it is increasingly difficult to screen for more than one desirable trait at a time, other methods of molecular evolution tend to be inhibitory. However, using recombination, it would be possible to combine the randomized fragments of the best representative variants for the various traits, and then select for multiple properties at once.

DNA shuffling can also be applied to the polynucleotides and polypeptides of the present invention to decrease their immunogenicity in a specified host. For example, a particular variant of the present invention may be created and isolated using DNA shuffling technology. Such a variant may have all of the desired characteristics, though may be highly immunogenic in a host due to its novel intrinsic structure. Specifically, the desired characteristic may cause the polypeptide to have a non-native structure which could no longer be recognized as a “self” molecule, but rather as a “foreign”, and thus activate a host immune response directed against the novel variant. Such a limitation can be overcome, for example, by including a copy of the gene sequence for a xenobiotic ortholog of the native protein in with the gene sequence of the novel variant gene in one or more cycles of DNA shuffling. The molar ratio of the ortholog and novel variant DNAs could be varied accordingly. Ideally, the resulting hybrid variant identified would contain at least some of the coding sequence which enabled the xenobiotic protein to evade the host immune system, and additionally, the coding sequence of the original novel variant that provided the desired characteristics.

Likewise, the invention encompasses the application of DNA shuffling technology to the evolution of polynucleotides and polypeptides of the invention, wherein one or more cycles of DNA shuffling include, in addition to the gene template DNA, oligonucleotides coding for known allelic sequences, optimized codon sequences, known variant sequences, known polynucleotide polymorphism sequences, known ortholog sequences, known homologue sequences, additional homologous sequences, additional non-homologous sequences, sequences from another species, and any number and combination of the above.

In addition to the described methods above, there are a number of related methods that may also be applicable, or desirable in certain cases. Representative among these are the methods discussed in PCT applications WO 98/31700, and WO 98/32845, which are hereby incorporated by reference. Furthermore, related methods can also be applied to the polynucleotide sequences of the present invention in order to evolve invention for creating ideal variants for use in gene therapy, protein engineering, evolution of whole cells containing the variant, or in the evolution of entire enzyme pathways containing polynucleotides of the invention as described in PCT applications WO 98/13485, WO 98/13487, WO 98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech., 15:436–438, (1997), respectively.

Additional methods of applying “DNA Shuffling” technology to the polynucleotides and polypeptides of the present invention, including their proposed applications, may be found in U.S. Pat. No. 5,605,793; PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCT Application No. WO 97/35966; and PCT Application No. WO 98/42832. PCT Application No. WO 00/09727 specifically provides methods for applying DNA shuffling to the identification of herbicide selective crops which could be applied to the polynucleotides and polypeptides of the present invention; additionally, PCT Application No. WO 00/12680 provides methods and compositions for generating, modifying, adapting, and optimizing polynucleotide sequences that confer detectable phenotypic properties on plant species; each of the above are hereby incorporated in their entirety herein for all purposes.

Example 21 Site Directed/Site-specific Mutagenesis

In vitro site-directed mutagenesis is an invaluable technique for studying protein structure-function relationships and gene expression, for example, as well as for vector modification. Site-directed mutagenesis can also be used for creating any of one or more of the mutants of the present invention, particularly the conservative and/or non-conservative amino acid substitution mutants of the prsent invention. Approaches utilizing single stranded DNA (ssDNA) as the template have been reported (e.g., T. A. Kunkel et al., 1985, Proc. Natl. Acad. Sci. USA), 82:488–492; M. A. Vandeyar et al., 1988, Gene, 65(1):129–133; M. Sugimoto et al., 1989, Anal. Biochem., 179(2):309–311; and J. W. Taylor et al., 1985, Nuc. Acids. Res., 13(24):8765–8785).

The use of PCR in site-directed mutagenesis accomplishes strand separation by using a denaturing step to separate the complementary strands and to allow efficient polymerization of the PCR primers. PCR site-directed mutagenesis methods thus permit site specific mutations to be incorporated in virtually any double stranded plasmid, thus eliminating the need for re-subcloning into M13-based bacteriophage vectors or single-stranded rescue. (M. P. Weiner et al., 1995, Molecular Biology: Current Innovations and Future Trends, Eds. A. M. Griffin and H. G. Griffin, Horizon Scientific Press, Norfolk, UK; and C. Papworth et al., 1996, Strategies, 9(3):3–4).

A protocol for performing site-directed mutagenesis, particularly employing the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, Calif.; U.S. Pat. Nos. 5,789,166 and 5,923,419) is provided for making point mutations, to switch or substitute amino acids, and to delete or insert single or multiple amino acids in the RATL1d6 amino acid sequence of this invention.

Primer Design

For primer design using this protocol, the mutagenic oligonucleotide primers are designed individually according to the desired mutation. The following considerations should be made for designing mutagenic primers: 1) Both of the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid; 2) Primers should be between 25 and 45 bases in length, and the melting temperature (T_(m)) of the primers should be greater than, or equal to, 78° C. The following formula is commonly used for estimating the T_(m) of primers: T=81.5+0.41 (% GC)−675/N−% mismatch. For calculating T_(m), N is the primer length in bases; and values for % GC and % mismatch are whole numbers. For calculating T_(m) for primers intended to introduce insertions or deletions, a modified version of the above formula is employed: T=81.5+0.41 (% GC)−675/N, where N does not include the, bases which are being inserted or deleted; 3) The desired mutation (deletion or insertion) should be in the middle of the primer with approximately 10–15 bases of correct sequence on both sides; 4) The primers optimally should have a minimum GC content of 40%, and should terminate in one or more C or G bases; 5) Primers need not be 5′-phosphorylated, but must be purified either by fast polynucleotide liquid chromatography (FPLC) or by polyacrylamide gel electrophoresis (PAGE). Failure to purify the primers results in a significant decrease in mutation efficiency; and 6) It is important that primer concentration is in excess. It is suggested to vary the amount of template while keeping the concentration of the primers constantly in excess (QuikChange™ Site-Directed Mutagenesis Kit, Stratagene, La Jolla, Calif.).

Protocol for Setting Up the Reactions

Using the above-described primer design, two complimentary oligonucleotides containing the desired mutation, flaniked by unmodified nucleic acid sequence, are synthesized. The resulting oligonucleotide primers are purified.

A control reaction is prepared using 5 μl 10× reaction buffer (100 mM KCl; 100 mM (NH₄)₂SO₄; 200 mM Tris-HCl, pH 8.8; 20 mM MgSO₄; 1% Triton® X-100; 1 mg/ml nuclease-free bovine serum albumin, BSA); 2 μl (10 ng) of pWhitescript™, 4.5-kb control plasmid (5 ng/μl); 1.25 μl (125 ng) of oligonucleotide control primer #1 (34-mer, 100 ng/μl); 1.25 μl (125 ng) of oligonucleotide control primer #2 (34-mer, 100 ng/μl); 1 μl of dNTP mix; double distilled H₂O; to a final volume of 50 μl. Thereafter, 1 μl of DNA polymerase (PfuTurbo® DNA Polymerase, Stratagene), (2.5U/μl) is added. PfuTurbo® DNA Polymerase is stated to have 6-fold higher fidelity in DNA synthesis than does Taq polymerase. To maximize temperature cycling performance, use of thin-walled test tubes is suggested to ensure optimum contact with the heating blocks of the temperature cycler.

The sample reaction is prepared by combining 5 μl of 10× reaction buffer; × μl (5–50 ng) of dsDNA template; × μl (125 ng) of oligonucleotide primer #1; × μl (5–50 ng) of dsDNA template; × μl (125 ng) of oligonucleotide primer #2; 1 μl of dNTP mix; and ddH₂O to a final volume of 50 μl. Thereafter, 1 μl of DNA polymerase (PfuTurbo DNA Polymerase, Stratagene), (2.5U/μl) is added.

It is suggested that if the thermal cycler does not have a hot-top assembly, each reaction should be overlaid with approximately 30 μl of mineral oil.

Cycling the Reactions

Each reaction is cycled using the following cycling parameters:

Segment Cycles Temperature Time 1 1 95° C. 30 seconds 2 12–18 95° C. 30 seconds 55° C.  1 minute 68° C.  2 minutes/kb of plasmid length

For the control reaction, a 12-minute extension time is used and the reaction is run for 12 cycles. Segment 2 of the above cycling parameters is adjusted in accordance with the type of mutation desired. For example, for point mutations, 12 cycles are used; for single amino acid changes, 16 cycles are used; and for multiple amino acid deletions or insertions, 18 cycles are used. Following the temperature cycling, the reaction is placed on ice for 2 minutes to cool the reaction to ≦37° C.

Digesting the Products and Transforming Competent Cells

One μl of the DpnI restriction enzyme (10U/μl) is added directly (below mineral oil overlay) to each amplification reaction using a small, pointed pipette tip. The reaction mixture is gently and thoroughly mixed by pipetting the solution up and down several times. The reaction mixture is then centrifuged for 1 minute in a microcentrifuge. Immediately thereafter, each reaction is incubated at 37° C. for 1 hour to digest the parental (i.e., the non-mutated) supercoiled dsDNA.

Competent cells (i.e., XL1-Blue supercompetent cells, Stratagene) are thawed gently on ice. For each control and sample reaction to be transformed, 50 μl of the supercompetent cells are aliquotted to a prechilled test tube (Falcon 2059 polypropylene). Next, 1 μl of the DpnI-digested DNA is transferred from the control and the sample reactions to separate aliquots of the supercompetent cells. The transformation reactions are gently swirled to mix and incubated for 30 minutes on ice. Thereafter, the transformation reactions are heat-pulsed for 45 seconds at 42° C. for 2 minutes.

0.5 ml of NZY+ broth, preheated to 42° C. is added to the transformation reactions which are then incubated at 37° C. for 1 hour with shaking at 225–250 rpm. An aliquot of each transformation reaction is plated on agar plates containing the appropriate antibiotic for the vector. For the mutagenesis and transformation controls, cells are spread on LB-ampicillin agar plates containing 80 μg/ml of X-gal and 20 mM IPTG. Transformation plates are incubated for >16 hours at 37° C.

Example 22 Method of Determining Alterations in a Gene Corresponding to a Polynucleotide

RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is be isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO:1. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60–120 seconds at 52–58 degrees C.; and 60–120 seconds at 70 degrees C., using buffer solutions described in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations is then cloned and sequenced to validate the results of the direct sequencing.

PCR products are cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to the methods described herein are nick-translated with digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73–99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991).) Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation. Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.

Example 23 Alternative Methods of Detecting Polymorphisms Encompassed by the Present Invention

A. Preparation of Samples

Polymorphisms are detected in a target nucleic acid from an individual being analyzed. For assay of genomic DNA, virtually any biological sample (other than pure red blood cells) is suitable. For example, convenient tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. For assay of cDNA or mRNA, the tissue sample must be obtained from an organ in which the target nucleic acid is expressed. For example, if the target nucleic acid is a cytochrome P450, the liver is a suitable source.

Many of the methods described below require amplification of DNA from target samples. This can be accomplished by e.g., PCR. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202.

Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4:560 (1989), Landegren et al., Science 241:1077 (1988), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989), and self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990)) and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.

Additional methods of amplification are known in the art or are described elsewhere herein.

B. Detection of Polymorphisms in Target DNA

There are two distinct types of analysis of target DNA for detecting polymorphisms. The first type of analysis, sometimes referred to as de novo characterization, is carried out to identify polymorphic sites not previously characterized (i.e., to identify new polymorphisms). This analysis compares target sequences in different individuals to identify points of variation, i.e., polymorphic sites. By analyzing groups of individuals representing the greatest ethnic diversity among humans and greatest breed and species variety in plants and animals, patterns characteristic of the most common alleles/haplotypes of the locus can be identified, and the frequencies of such alleles/haplotypes in the population can be determined. Additional allelic frequencies can be determined for subpopulations characterized by criteria such as geography, race, or gender. The de novo identification of polymorphisms of the invention is described in the Examples section.

The second type of analysis determines which form(s) of a characterized (known) polymorphism are present in individuals under test. Additional methods of analysis are known in the art or are described elsewhere herein.

1. Allele-specific Probes

The design and use of allele-specific probes for analyzing polymorphisms is described by e.g., Saiki et al., Nature 324,163–166 (1986); Dattagupta, E P 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the corresponding segment from another individual due to the presence of different polymorphic forms in the respective segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e.g., in a 15-mer at the 7 position; in a 16-mer, at either the 8 or 9 position) of the probe. This design of probe achieves good discrimination in hybridization between different allelic forms.

Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target sequence.

2. Tiling Arrays

The polymorphisms can also be identified by hybridization to nucleic acid arrays, some examples of which are described in WO 95/11995. The same arrays or different arrays can be used for analysis of characterized polymorphisms. -WO 95/11995 also describes sub arrays that are optimized for detection of a variant form of a precharacterized polymorphism. Such a sub array contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence. The second group of probes is designed by the same principles as described, except that the probes exhibit complementarity to the second reference sequence. The inclusion of a second group (or further groups) can be particularly useful for analyzing short subsequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length of the probes (e.g., two or more mutations within 9 to bases).

3. Allele-Specific Primers

An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17,2427–2448 (1989). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers. resulting in a detectable product which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed. The method works best when the mismatch is included in the 3′-most position of the oligonucleotide aligned with the polymorphism because this position is most destabilizing elongation from the primer (see, e.g., WO 93/22456).

4. Direct-Sequencing

The direct analysis of the sequence of polymorphisms of the present invention can be accomplished using either the dideoxy chain termination method or the Maxam-Gilbert method (see Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).

5. Denaturing Gradient Gel Electrophoresis

Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Erlich, ed., PCR Technology. Principles and Applications for DNA Amplification, (W. H. Freeman and Co, New York, 1992), Chapter 7.

6. Single-Strand Conformation Polymorphism Analysis

Alleles of target sequences can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al., Proc. Nat. Acad. Sci. 86,2766–2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence. The different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences.

7. Single Base Extension

An alternative method for identifying and analyzing polymorphisms is based on single-base extension (SBE) of a fluorescently-labeled primer coupled with fluorescence resonance energy transfer (FRET) between the label of the added base and the label of the primer. Typically, the method, such as that described by Chen et al., (PNAS 94:10756–61 (1997), uses a locus-specific oligonucleotide primer labeled on the 5′ terminus with 5-carboxyfluorescein (F AM). This labeled primer is designed so that the 3′ end is immediately adjacent to the polymorphic site of interest. The labeled primer is hybridized to the locus, and single base extension of the labeled primer is performed with fluorescently-labeled dideoxyribonucleotides (ddNTPs) in dye-terminator sequencing fashion. An increase in fluorescence of the added ddNTP in response to excitation at the wavelength of the labeled primer is used to infer the identity of the added nucleotide.

Example 24 Method of Genotyping Each SNP of the Present Invention

a.) Genomic DNA Preparation

Genomic DNA samples for genotyping may be prepared using the Purigene™ DNA extraction kit from Gentra Systems. After preparation, DNA samples may be diluted to a 2 ng/ul working concentration with TE buffer (10 mM Tris-Cl, pH 8.0, 0.1 mM EDTA, pH 8.0) and stored in 1 ml 96 deep well plates (VWR) at −20 degrees until use.

Samples for genomic DNA preparation may be obtained from the Coriell Institute (Collingswood, N.J.), patients participating in a Bristol-Myers Squibb (BMS) clinical study, or from other sources known in the art or otherwise described herein.

b) Genotyping

The SNP genotyping reactions may be performed using the SNPStream™ system (Orchid Biosience, Princeton, N.J.) based on genetic bit analysis (Nikiforov, T. et al, Nucleic Acids Res 22, 4167–4175 (1994)).

The regions including polymorphic sites may be amplified by the polymerase chain reaction (PCR) using a pair of primers (OPERON Technologies), one of which can be phosphorothioated. 6 ul PCR cocktail containing 1.0 ng/ul genomic DNA, 200 uM dNTPs, 0.5 uM forward PCR primer, 0.5 uM reverse PCR primer (phosphorothioated), 0.05 u/ul Platinum Taq DNA polymerase (LifeTechnologies), and 1.5 mM MgCl₂. The PCR primer pairs used for genotyping analysis can be designed using methods known in the art in conjunction with the teachings of the present invention. The PCR reaction can be set up in 384-well plates (MJ Research) using a MiniTrak liquid handling station (Packard Bioscience). PCR thermocycling can be performed under the following conditions in a MJ Research Tetrad machine: step1, 95 degrees for 2 min; step 2, 94 degrees for 30 min; step 3, 55 degrees for 2 min; step 4, 72 degrees for 30 sec; step 5, go back to step 2 for an additional 39 cycles; step 6, 72 degrees for 1 min; and step 7, 12 degrees indefinitely)

After thermocycling, the amplified samples may be placed in the SNPStream™ (Orchid Bioscience) machine, and automated genetic bit analysis (GBA) (Nikiforov, T. et al,supra) reaction can be performed. The first step of this reaction can be degradation of one of the strands of the PCR products by T7 gene 6 exonuclease to make them single-stranded. The strand containing phosphorothioated primer are resistant to T7 gene 6 nuclease, and may be not degraded by this enzyme. After digestion, the single-stranded PCR products may be subjected to an annealing step whereby the single stranded PCR products may be annealed to the GBA primer on a solid phase, and then subjected to the GBA reaction (single base extension) using dideoxy-NTPs labeled with biotin or fluorescein. The GBA primers may be designed using methods known in the art in conjunction with the teachings of the present invention. The present invention encompasses the substitution of certain polynucleotides within the GBA primers with a polynucleotide that may be substituted with a C3 linker (C3 spacer phosphoramidite) during synthesis of the primer. Such linkers may be obtained from Research Genetics, and Sigma-Genosys, for example. Such primers may be obtained from Operon. Incorporation of these dideoxynucleotides into a GBA primer may be detected by two color ELISA assay using anti-fluorescein alkaline phosphatase conjugate and anti-biotin horseradish peroxidase. Automated genotype calls may be made by GenoPak software (Orchid Bioscience), before manual correction of automated calls may be done upon inspection of the resulting allelogram of each SNP.

Example 25 Alternative Method of Genotyping Each SNP of the Present Invention

In addition to the method of genotyping described herein, the skilled artisan could determine the genotype of the polymorphisms of the present invention using the below described alternative method. This method is referred to as the “GBS method” herein and may be performed as described in conjunction with the teaches described elsewhere herein.

Briefly, the direct analysis of the sequence of the polymorphisms of the present invention can be accomplished by DNA sequencing of PCR products corresponding to the same. PCR amplicons are designed to be in close proximity to the polymorphisms of the present invention using the Primer3 program. The M13_SEQUENCE1 “TGTAAAACGACGGCCAGT (SEQ ID NO:71)” is prepended to each forward PCR primer. The M13_SEQUENCE2 “CAGGAAACAGCTATGACC (SEQ ID NO:72)” is prepended to each reverse PCR primer.

PCR amplification can be performed on genomic DNA samples amplified from (20 ng) in reactions (50 ul) containing 10 mM Tris-Cl pH 8.3, 50 mM KCl, 2.5 mM MgCl₂, 150 uM dNTPs, 3 uM PCR primers, and 3.75 U TaqGold DNA polymerase (PE Biosystems). PCR can be performed in MJ Research Tetrad machines under a cycling condition of 94 degrees 10 min, 30 cycles of 94 degrees 30 sec, 60 degrees 30 sec, and 72 degrees 30 sec, followed by 72 degrees 7 min. PCR products may be purified using QIAquick PCR purification kit (Qiagen), and may be sequenced by the dye-terminator method using PRISM 3700 automated DNA sequencer (Applied Biosystems, Foster City, Calif.) following the manufacturer's instruction outlined in the Owner's Manual (which is hereby incorporated herein by reference in its entirety).essentially the same as described in herein.

PCR products are sequenced by the dye-terminator method using the M13_SEQUENCE1 (SEQ ID NO:X) and M13_-SEQUENCE2 (SEQ ID NO:X) primers above. The genotype can be determined by analysis of the sequencing results at the polymorphic position.

Example 26 Additional Methods of Genotyping the SNPs of the Present Invention

The skilled artisan would acknowledge that there are a number of methods that may be employed for genotyping a SNP of the present invention, aside from the preferred methods described herein. The present invention encompasses the following non-limiting types of genotype assays: PCR-free genotyping methods, Single-step homogeneous methods, Homogeneous detection with fluorescence polarization, Pyrosequencing, “Tag” based DNA chip system, Bead-based methods, fluorescent dye chemistry, Mass spectrometry based genotyping assays, TaqMan genotype assays, Invader genotype assays, and microfluidic genotype assays, among others.

Specifically encompassed by the present invention are the following, non-limiting genotyping methods: Landegren, U., Nilsson, M. & Kwok, P. 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Example 27 Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample

A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described elsewhere herein. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbounded polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25–400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide in the sample using the standard curve.

Example 28 Formulation

The invention also provides methods of treatment and/or prevention diseases, disorders, and/or conditions (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant a polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Therapeutic alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount of the Therapeutic administered parenterally per dose will be in the range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the Therapeutic is typically administered at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1–4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.

Therapeutics can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

In yet an additional embodiment, the Therapeutics of the invention are delivered orally using the drug delivery technology described in U.S. Pat. No. 6,258,789. which is hereby incorporated by reference herein.

Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Therapeutics of the invention may also be suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547–556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167–277 (1981), and Langer, Chem. Tech. 12:98–105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release Therapeutics also include liposomally entrapped Therapeutics of the invention (see, generally, Langer, Science 249:1527–1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317–327 and 353–365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688–3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.(USA) 77:4030–4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200–800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.

In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527–1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polimers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic will typically be formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1–10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the Therapeutics of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeutics of the invention are administered in combination with alum. In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the Therapeutics of the invention, include but not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines and/or growth factors. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.

In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors. Nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIR (zidovudine/AZT), VIDEX (didanosine/ddI), HIVID (zalcitabine/ddC), ZERIT (stavudine/d4T), EPIVIR (lamivudine/3TC), and COMBIVIR (zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to. VIRAMUNE (nevirapine), RESCRIPTOR (delavirdine), and SUSTIVA (efavirenz). Protease inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, CRIXIVAN (indinavir), NORVIR (ritonavir), INVIRASE (saquinavir), and VIRACEPT (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV infection.

In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, ATOVAQUONE, ISONIAZID, RIFAMPIN, PYRAZINAMIDE, ETHAMBUTOL, RIFABUTIN, CLARITHROMYCIN, AZITHROMYCIN, GANCICLOVIR, FOSCARNET, CIDOFOVIR, FLUCONAZOLE, ITRACONAZOLE, KETOCONAZOLE, ACYCLOVIR, FAMCICOLVIR, PYRIMETHAMINE, LEUCOVORIN, NEUPOGEN (filgrastim/G-CSF), and LEUKINE (sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, and/or ATOVAQUONE to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONIAZID, RIFAMPIN, PYRAZINAMIDE, and/or ETHAMBUTOL to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTIN, CLARITHROMYCIN, and/or AZITHROMYCIN to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIR, FOSCARNET, and/or CIDOFOVIR to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLE, ITRACONAZOLE, and/or KETOCONAZOLE to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIR and/or FAMCICOLVIR to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINE and/or LEUCOVORIN to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with LEUCOVORIN and/or NEUPOGEN to prophylactically treat or prevent an opportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.

In a further embodiment, the Therapeutics of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the Therapeutics of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.

In specific embodiments, Therapeutics of the invention are administered in combination with immunosuppressants. Immunosuppressants preparations that may be administered with the Therapeutics of the invention include, but are not limited to, ORTHOCLONE (OKT3), SANDIMMUNE/NEORAL/SANGDYA (cyclosporin), PROGRAF (tacrolimus), CELLCEPT (mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMAR, IVEEGAM, SANDOGLOBULIN, GAMMAGARD S/D, and GAMIMUNE. In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the Therapeutics of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

In another embodiment, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g. tamoxifen); antimetabolites (e.g. fluorouracil, 5-FU. methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, Therapeutics of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or any combination of the components of CHOP. In another embodiment, Therapeutics of the invention are administered in combination with Rituximab. In a further embodiment, Therapeutics of the invention are administered with Rituxmab and CHOP, or Rituxmab and any combination of the components of CHOP.

In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines. Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, Therapeutics of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In an additional embodiment, the Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816: Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PIGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (P1GF-2), as disclosed in Hauser et al., Growth Factors, 4:259–268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are incorporated herein by reference herein.

In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, LEUKINE (SARGRAMOSTIM) and NEUPOGEN (FILGRASTIM).

In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

In a specific embodiment, formulations of the present invention may further comprise antagonists of P-glycoprotein (also referred to as the multiresistance protein, or PGP), including antagonists of its encoding polynucleotides (e.g., antisense oligonucleotides, ribozymes, zinc-finger proteins, etc.). P-glycoprotein is well known for decreasing the efficacy of various drug administrations due to its ability to export intracellular levels of absorbed drug to the cell exterior. While this activity has been particularly pronounced in cancer cells in response to the administration of chemotherapy regimens, a variety of other cell types and the administration of other drug classes have been noted (e.g., T-cells and anti-HIV drugs). In fact, certain mutations in the PGP gene significantly reduces PGP function, making it less able to force drugs out of cells. People who have two versions of the mutated gene—one inherited from each parent—have more than four times less PGP than those with two normal versions of the gene. People may also have one normal gene and one mutated one. Certain ethnic populations have increased incidence of such PGP mutations. Among individuals from Ghana, Kenya, the Sudan, as well as African Americans, frequency of the normal gene ranged from 73% to 84%. In contrast, the frequency was 34% to 59% among British whites, Portuguese, Southwest Asian, Chinese, Filipino and Saudi populations. As a result, certain ethnic populations may require increased administration of PGP antagonist in the formulation of the present invention to arrive at the an efficacious dose of the therapeutic (e.g., those from African descent). Conversely, certain ethnic populations, particularly those having increased frequency of the mutated PGP (e.g., of Caucasian descent, or non-African descent) may require less pharmaceutical compositions in the formulation due to an effective increase in efficacy of such compositions as a result of the increased effective absorption (e.g., less PGP activity) of said composition.

Moreover, in another specific embodiment, formulations of the present invention may further comprise antagonists of OATP2 (also referred to as the multiresistance protein, or MRP2), including antagonists of its encoding polynucleotides (e.g., antisense oligonucleotides, ribozymes, zinc-finger proteins, etc.). The invention also further comprises any additional antagonists known to inhibit proteins thought to be attributable to a multidrug resistant phenotype in proliferating cells.

Preferred antagonists that formulations of the present may comprise include the potent P-glycoprotein inhibitor elacridar, and/or LY-335979. Other P-glycoprotein inhibitors known in the art are also encompassed by the present invention.

In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

Example 29 Method of Treating Decreased Levels of the Polypeptide

The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the invention (including polypeptides of the invention). Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the polypeptide to increase the activity level of the polypeptide in such an individual.

For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1–100 ug/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided herein.

Example 30 Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer. For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided herein.

Example 31 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C. for approximately one week.

At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219–25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.

The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 9 using primers and having appropriate restriction sites and initiation/stop codons. if necessary. Preferably the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.

The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.

Example 32 Gene Therapy Using Endogenous Genes Corresponding to Polynucleotides of the Invention

Another method of gene therapy according to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous recombination as described, for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932–8935 (1989); and Zijlstra et al., Nature, 342:435–438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5′ non-coding sequence of endogenous polynucleotide sequence, flanking the promoter. The targeting sequence will be sufficiently near the 5′ end of the polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The construct is size fractionated on an agarose gel then purified by phenol extraction and ethanol precipitation.

In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-facilitating agents, such as liposomes, viral sequences, viral particles. precipitating agents, etc. Such methods of delivery are known in the art.

Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results in the expression of polynucleotide corresponding to the polynucleotide in the cell. Expression may be detected by immunological staining, or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM+10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contains approximately 3×106 cells/ml. Electroporation should be performed immediately following resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII The CMV promoter is amplified by PCR with an XbaI site on the 5′ end and a BamHI. site on the 3′end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5′ end and an Xba site at the 3′end; the other non-coding sequence (fragment 2) is amplified with a BamHI site at the 5′end and a HindIII site at the 3′end. The CMV promoter and the fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI; fragment 2—BamHI) and ligated together. The resulting ligation product is digested with HindIII, and ligated with the HindIII-digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×106 cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and 250–300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14–20 mSec should be observed.

Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16–24 hours.

The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 33 Method of Treatment Using Gene Therapy—in Vivo

Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470–479 (1997); Chao et al., Pharmacol. Res. 35(6):517–522 (1997) Wolff, Neuromuscul. Disord. 7(5):314–318 (1997); Schwartz et al., Gene Ther. 3(5):405–411 (1996); Tsurumi et al., Circulation 94(12):3281–3290 (1996) (incorporated herein by reference).

The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126–139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1–7) which can be prepared by methods well known to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapies techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be use to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.

Example 34 Transgenic Animals

The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.

Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691–698 (1994); Carver et al., Biotechnology (NY) 11: 1263–1270 (1993); Wright et al., Biotechnology (NY) 9:830–834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148–6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313–321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803–1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717–723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171–229 (1989), which is incorporated by reference herein in its entirety.

Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64–66 (1996); Wilmut et al., Nature 385:810–813 (1997)).

The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232–6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103–106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR(RT-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.

Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 35 Knock-out Animals

Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (E.g., see Smithies et al., Nature 317:230–234 (1985); Thomas & Capecchi, Cell 51:503–512 (1987); Thompson et al., Cell 5:313–321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.

In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 36 Method of Isolating Antibody Fragments Directed Against MMP-29 from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against MMP-29 to which the donor may or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in its entirety).

Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in PCT publication WO 92/01047. To rescue phage displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to inoculate 50 ml of 2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, see PCT publication WO 92/01047) are added and the culture incubated at 37° C. for 45 minutes without shaking and then at 37° C. for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are prepared as described in PCT publication WO 92/01047.

M13 delta gene III is prepared as-follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37° C. without shaking and then for a further hour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 1013 transducing units/ml (ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0 M Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37° C. The E. coli are then plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtitre plates coated with either 10 pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., PCT publication WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.

Moreover, in another preferred method, the antibodies directed against the polypeptides of the present invention may be produced in plants. Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and 6,080,560, which are hereby incorporated in their entirety herein. The methods not only describe methods of expressing antibodies, but also the means of assembling foreign multimeric proteins in plants (i.e., antibodies, etc,), and the subsequent secretion of such antibodies from the plant.

Example 37 Identification and Cloning of VH and VL Domains of Antibodies Directed Against the MMP-29 Polypeptide

VH and VL domains may be identified and cloned from cell lines expressing an antibody directed against a MMP-29 epitope by performing PCR with VH and VL specific primers on cDNA made from the antibody expressing cell lines. Briefly, RNA is isolated from the cell lines and used as a template for RT-PCR designed to amplify the VH and VL domains of the antibodies expressed by the EBV cell lines. Cells may be lysed using the TRIzol reagent (Life Technologies, Rockville, Md.) and extracted with one fifth volume of chloroform. After addition of chloroform, the solution is allowed to incubate at room temperature for 10 minutes, and then centrifuged at 14,000 rpm for 15 minutes at 4 C in a tabletop centrifuge. The supernatant is collected and RNA is precipitated using an equal volume of isopropanol. Precipitated RNA is pelleted by centrifuging at 14,000 rpm for 15 minutes at 4 C in a tabletop centrifuge.

Following centrifugation, the supernatant is discarded and washed with 75% ethanol. Following the wash step, the RNA is centrifuged again at 800 rpm for 5 minutes at 4 C. The supernatant is discarded and the pellet allowed to air dry. RNA is the dissolved in DEPC water and heated to 60 C for 10 minutes. Quantities of RNA can be determined using optical density measurements. cDNA may be synthesized, according to methods well-known in the art and/or described herein, from 1.5–2.5 micrograms of RNA using reverse transciptase and random hexamer primers. cDNA is then used as a template for PCR amplification of VH and VL domains.

Primers used to amplify VH and VL genes are shown below. Typically a PCR reaction makes use of a single 5′ primer and a single 3′ primer. Sometimes, when the amount of available RNA template is limiting, or for greater efficiency, groups of 5′ and/or 3′ primers may be used. For example, sometimes all five VH-5′ primers and all JH3′ primers are used in a single PCR reaction. The PCR reaction is carried out in a 50 microliter volume containing 1×PCR buffer, 2 mM of each dNTP, 0.7 units of High Fidelity Taq polymerse, 5′ primer mix, 3′ primer mix and 7.5 microliters of cDNA. The 5′ and 3′ primer mix of both VH and VL can be made by pooling together 22 pmole and 28 pmole, respectively, of each of the individual primers. PCR conditions are : 96 C for 5 minutes; followed by 25 cycles of 94 C for 1 minute, 50 C for 1 minute, and 72 C for 1 minute; followed by an extension cycle of 72 C for 10 minutes. After the reaction has been completed, sample tubes may be stored at 4 C.

SEQ ID Primer name Primer Sequence NO: Primer Sequences Used to Amplify VH domains. Hu VH1-5′ CAGGTGCAGCTGGTGCAGTCTGG 32 Hu VH2-5′ CAGGTCAACTTAAGGGAGTCTGG 33 Hu VH3-5′ GAGGTGCAGCTGGTGGAGTCTGG 34 Hu VH4-5′ CAGGTGCAGCTGCAGGAGTCGGG 35 Hu VHS-5′ GAGGTGCAGCTGTTGCAGTCTGC 36 Hu VH6-5′ CAGGTACAGCTGCAGCAGTCAGG 37 Hu JH1-5′ TGAGGAGACGGTGACCAGGGTGCC 38 Hu JH3-5′ TGAAGAGACGGTGACCATTGTCCC 39 Hu JH4-5′ TGAGGAGACGGTGACCAGGGTTCC 40 Hu JH6-5′ TGAGGAGACGGTGACCGTGGTCCC 41 Primer Sequences Used to Amplify VL domains Hu Vkappa1-5′ GACATCCAGATGACCCAGTCTCC 42 Hu Vkappa2a-5′ GATGTTGTGATGACTCAGTCTCC 43 Hu Vkappa2b-5′ GATATTGTGATGACTCAGTCTCC 44 Hu Vkappa3-5′ GAAATTGTGTTGACGCAGTCTCC 45 Hu Vkappa4-5′ GACATCGTGATGACCCAGTCTCC 46 Hu Vkappa5-5′ GAAACGACACTCACGCAGTCTCC 47 Hu Vkappa6-5′ GAAATTGTGCTGACTCAGTCTCC 48 Hu Vlambda1-5′ CAGTCTGTGTTGACGCAGCCGCC 49 Hu Vlambda2-5′ CAGTCTGCCCTGACTCAGCCTGC 50 Hu Vlambda3-5′ TCCTATGTGCTGACTCAGCCACC 51 Hu Vlambda3b-5′ TCTTCTGAGCTGACTCAGGACCC 52 Hu Vlambda4-5′ CACGTTATACTGACTCAACCGCC 53 Hu Vlambda5-5′ CAGGCTGTGCTCACTCAGCCGTC 54 Hu Vlambda6-5′ AATTTTATGCTGACTCAGCCCCA 55 Hu Jkappal-3′ ACGTTTGATTTCCACCTTGGTCCC 56 Hu Jkappa2-3′ ACGTTTGATCTCCAGCTTGGTCCC 57 Hu Jkappa3-3′ ACGTTTGATATCCACTTTGGTCCC 58 Hu Jkappa4-3′ ACGTTTGATCTCCACCTTGGTCCC 59 Hu Jkappa5-3′ ACGTTTAATCTCCAGTCGTGTCCC 60 Hu Vlambdal-3′ CAGTCTGTGTTGACGCAGCCGCC 61 Hu Vlambda2-3′ CAGTCTGCCCTGACTCAGCCTGC 62 Hu Vlambda3-3′ TCCTATGTGCTGACTCAGCCACC 63 Hu Vlambda3b-3′ TCTTCTGAGCTGACTCAGGACCC 64 Hu Vlambda4-3′ CACGTTATACTGACTCAACCGCC 65 Hu Vlambda5-3′ CAGGCTGTGCTCACTCAGCCGTC 66 Hu Vlambda6-3′ AATTTTATGCTGACTCAGCCCCA 67

PCR samples are then electrophoresed on a 1.3% agarose gel. DNA bands of the expected sizes (−506 base pairs for VH domains, and 344 base pairs for VL domains) can be cut out of the gel and purified using methods well known in the art and/or described herein.

Purified PCR products can be ligated into a PCR cloning vector (TA vector from Invitrogen Inc., Carlsbad, Calif.). Individual cloned PCR products can be isolated after transfection of E. coli and blue/white color selection. Cloned PCR products may then be sequenced using methods commonly known in the art and/or described herein.

The PCR bands containing the VH domain and the VL domains can also be used to create full-length Ig expression vectors. VH and VL domains can be cloned into vectors containing the nucleotide sequences of a heavy (e. g., human IgGI or human IgG4) or light chain (human kappa or human ambda) constant regions such that a complete heavy or light chain molecule could be expressed from these vectors when transfected into an appropriate host cell. Further, when cloned heavy and light chains are both expressed in one cell line (from either one or two vectors), they can assemble into a complete functional antibody molecule that is secreted into the cell culture medium. Methods using polynucleotides encoding VH and VL antibody domain to generate expression vectors that encode complete antibody molecules are well known within the art.

Example 38 Biological Effects of Polypeptides of the Invention

Astrocyte and Neuronal Assays.

Recombinant polypeptides of the invention, expressed in Escherichia coli and purified as described above, can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate a polypeptide of the invention's activity on these cells.

Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.” Proc. Natl. Acad. Sci. USA 83:3012–3016. (1986), assay herein incorporated by reference in its entirety). However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of a polypeptide of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.

Fibroblast and Endothelial Cell Assays.

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CytoFluor fluorescence reader. For the PGE2 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium. the cells are incubated with FGF-2 or polypeptides of the invention with or without IL-1 (for 24 hours. The supernatants are collected and assayed for PGE2 by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without polypeptides of the invention IL-1(for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or polypeptides of the invention for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10–2500 ng/ml which can be used to compare stimulation with polypeptides of the invention.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson's that has been extensively characterized involves the systemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released. Subsequently, MPP+ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP+ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, polypeptides of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential effect of a polypeptide of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm2 on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N1). The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopaminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro. Therefore, if a polypeptide of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the polypeptide may be involved in Parkinson's Disease.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 38 Stimulation of Endothelial Migration

This example will be used to explore the possibility that a polypeptide of the invention may stimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 well microchemotaxis chamber (Neuroprobe Inc., Cabin John, MD; Falk, W., et al., J. Immunological Methods 1980;33:239–247). Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. Test substances are diluted to appropriate concentrations in M199 supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of the final dilution is placed in the lower chamber of the modified Boyden apparatus. Subconfluent, early passage (2–6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between lower and upper chamber, 2.5×105 cells suspended in 50 ul M199 containing 1% FBS are seeded in the upper compartment. The apparatus is then incubated for 5 hours at 37° C. in a humidified chamber with 5% CO2 to allow cell migration. After the incubation period, the filter is removed and the upper side of the filter with the non-migrated cells is scraped with a rubber policeman. The filters are fixed with methanol and stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration is quantified by counting cells of three random high-power fields (40×) in each well, and all groups are performed in quadruplicate.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 40 Method for Chromosomal Mapping of MMP-29 in 2q32 Flanking a Region Candidate for the Juvenile Form of Amyotrophic Lateral Sclerosis (ALS2)

Chromosomal localization of the MMP-29 gene was assessed by using the nucleic acid sequence of the invention (SEQ ID NO:1) in a database search of the recently completed draft of human genome sequence. Using the Basic Local Alignment Search Tool 2 (BLAST2), the first 200 bp of MMP-29 cDNA sequence (SEQ ID NO:1) aligned perfectly with Homo sapiens chromosome 2 Bac clones RP11-88L20 (AC012488) and RP11-455J20 (AC013468). Although the mapping of Bac RP11-455J20 has not yet been established, Bac RP11-88L20 was already registered in the clone database (NCBI), and mapped by Washington University Genome center, to chromosome 2q32. The sequence of MMP-29 flanks on the centromeric side the 1.7cM critical candidate region for ALS2 between marker D2S116 and D2S2237 defined by linkage and haplotype analyses in one consanguineous Tunisian family, characterized by a loss of upper motor neurons and spasticity of limb and facial muscles accompanying distal amyotrophy of hands and feet (Hamida et al., 1990; Hosler et al., 1998). Several candidate transcripts have been characterized in this interval (which excludes MMP-29 as a candidate gene based on genetic analyses) but none so far have been characterized as the causative gene for ALS2. There are no other diseases mapped in this chromosomal area representing a “good candidate” for MMP-29. Thus, there is a significant likelihood that aberrations in MMP-29 may be directly, or indirectly, associated with the incidence of ALS2.

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties.

TABLE IV Atom Atom Residue No Name Residue No X coord Y coord Z coord 1 N ALA 48 −1.453 84.982 97.717 2 CA ALA 48 −0.751 85.431 96.497 3 C ALA 48 0.353 86.484 96.681 4 O ALA 48 1.441 86.276 96.133 5 CB ALA 48 −1.791 85.970 95.523 6 N ASP 49 0.210 87.396 97.635 7 CA ASP 49 1.260 88.406 97.872 8 C ASP 49 2.544 87.771 98.411 9 O ASP 49 3.632 87.980 97.859 10 CB ASP 49 0.787 89.416 98.922 11 CG ASP 49 −0.421 90.241 98.480 12 OD1 ASP 49 −0.667 90.318 97.285 13 OD2 ASP 49 −1.080 90.788 99.358 14 N LEU 50 2.373 86.809 99.300 15 CA LEU 50 3.515 86.184 99.960 16 C LEU 50 3.930 84.870 99.307 17 O LEU 50 5.013 84.357 99.600 18 CB LEU 50 3.192 86.002 101.432 19 CG LEU 50 1.771 85.516 101.660 20 CD1 LEU 50 1.776 84.149 102.317 21 CD2 LEU 50 0.978 86.508 102.503 22 N HIS 51 3.151 84.405 98.346 23 CA HIS 51 3.596 83.298 97.505 24 C HIS 51 4.442 83.879 96.382 25 O HIS 51 5.485 83.306 96.044 26 CB HIS 51 2.384 82.561 96.950 27 CG HIS 51 2.724 81.325 96.139 28 ND1 HIS 51 3.814 80.542 96.266 29 CD2 HIS 51 1.962 80.784 95.131 30 CE1 HIS 51 3.756 79.544 95.363 31 NE2 HIS 51 2.611 79.695 94.660 32 N ALA 52 4.168 85.135 96.064 33 CA ALA 52 5.037 85.890 95.168 34 C ALA 52 6.353 86.182 95.873 35 O ALA 52 7.404 85.825 95.332 36 CB ALA 52 4.352 87.203 94.808 37 N ALA 53 6.276 86.505 97.156 38 CA ALA 53 7.488 86.725 97.960 39 C ALA 53 8.274 85.440 98.229 40 O ALA 53 9.510 85.462 98.200 41 CB ALA 53 7.088 87.363 99.285 42 N GLN 54 7.582 84.315 98.241 43 CA GLN 54 8.243 83.021 98.378 44 C GLN 54 8.965 82.635 97.098 45 O GLN 54 10.146 82.278 97.167 46 CB GLN 54 7.187 81.975 98.675 47 CG GLN 54 7.768 80.572 98.607 48 CD GLN 54 6.631 79.591 98.817 49 OE1 GLN 54 6.334 78.750 97.962 50 NE2 GLN 54 5.923 79.805 99.908 51 N ARG 55 8.389 82.975 95.957 52 CA ARG 55 9.070 82.714 94.694 53 C ARG 55 10.219 83.688 94.477 54 O ARG 55 11.299 83.203 94.116 55 CB ARG 55 8.062 82.772 93.557 56 CG ARG 55 7.290 81.459 93.501 57 CD ARG 55 6.170 81.490 92.472 58 NE ARG 55 5.036 82.281 92.964 59 CZ ARG 55 3.928 82.478 92.249 60 NH1 ARG 55 3.884 82.087 90.973 61 NH2 ARG 55 2.909 83.160 92.774 62 N PHE 56 10.135 84.849 95.113 63 CA PHE 56 11.225 85.837 95.070 64 C PHE 56 12.451 85.381 95.864 65 O PHE 56 13.561 85.856 95.601 66 CB PHE 56 10.786 87.175 95.676 67 CG PHE 56 9.602 87.917 95.052 68 CD1 PHE 56 8.917 88.864 95.800 69 CD2 PHE 56 9.212 87.668 93.743 70 CE1 PHE 56 7.837 89.537 95.256 71 CE2 PHE 56 8.125 88.342 93.196 72 CZ PHE 56 7.436 89.278 93.956 73 N LEU 57 12.294 84.405 96.741 74 CA LEU 57 13.438 83.962 97.528 75 C LEU 57 13.823 82.502 97.253 76 O LEU 57 15.014 82.177 97.282 77 CB LEU 57 13.063 84.167 98.989 78 CG LEU 57 12.568 85.595 99.229 79 CD1 LEU 57 11.828 85.745 100.551 80 CD2 LEU 57 13.683 86.626 99.098 81 N SER 58 12.862 81.677 96.867 82 CA SER 58 13.143 80.249 96.646 83 C SER 58 13.276 79.859 95.175 84 O SER 58 13.801 78.782 94.870 85 CB SER 58 12.034 79.412 97.278 86 OG SER 58 10.836 79.615 96.538 87 N ARG 59 12.746 80.674 94.279 88 CA ARG 59 12.960 80.434 92.852 89 C ARG 59 13.977 81.425 92.330 90 O ARG 59 14.620 81.201 91.299 91 CB ARG 59 11.668 80.621 92.069 92 CG ARG 59 10.734 79.424 92.161 93 CD ARG 59 9.597 79.604 91.164 94 NE ARG 59 10.155 80.003 89.861 95 CZ ARG 59 9.541 80.844 89.027 96 NH1 ARG 59 8.293 81.239 89.280 97 NH2 ARG 59 10.141 81.208 87.891 98 N TYR 60 14.048 82.543 93.030 99 CA TYR 60 15.033 83.575 92.729 100 C TYR 60 16.179 83.408 93.723 101 O TYR 60 17.070 82.565 93.561 102 CB TYR 60 14.460 84.983 92.929 103 CG TYR 60 13.156 85.479 92.272 104 CD1 TYR 60 12.181 84.624 91.775 105 CD2 TYR 60 12.962 86.854 92.197 106 CE1 TYR 60 10.999 85.130 91.255 107 CE2 TYR 60 11.790 87.367 91.658 108 CZ TYR 60 10.804 86.501 91.205 109 OH TYR 60 9.599 87.006 90.768 110 N GLY 61 16.132 84.213 94.767 111 CA GLY 61 17.113 84.076 95.840 112 C GLY 61 18.186 85.152 95.829 113 O GLY 61 17.934 86.297 96.219 114 N TRP 62 19.409 84.727 95.559 115 CA TRP 62 20.533 85.666 95.613 116 C TRP 62 21.557 85.438 94.501 117 O TRP 62 22.089 86.409 93.946 118 CB TRP 62 21.196 85.600 96.990 119 CG TRP 62 21.984 84.346 97.344 120 CD1 TRP 62 21.526 83.050 97.430 121 CD2 TRP 62 23.387 84.305 97.672 122 NE1 TRP 62 22.566 82.246 97.759 123 CE2 TRP 62 23.698 82.956 97.913 124 CE3 TRP 62 24.373 85.278 97.763 125 CZ2 TRP 62 25.000 82.600 98.240 126 CZ3 TRP 62 25.672 84.910 98.090 127 CH2 TRP 62 25.984 83.576 98.327 128 N SER 63 21.754 84.187 94.118 129 CA SER 63 22.770 83.852 93.117 130 C SER 63 22.898 82.351 92.955 131 O SER 63 22.183 81.560 93.583 132 CB SER 63 24.128 84.350 93.593 133 OG SER 63 24.408 83.650 94.796 134 N GLY 64 23.834 81.984 92.098 135 CA GLY 64 24.219 80.583 91.951 136 C GLY 64 25.021 80.153 93.177 137 O GLY 64 24.419 79.809 94.201 138 N VAL 65 26.330 80.367 93.086 139 CA VAL 65 27.407 79.980 94.044 140 C VAL 65 27.067 79.241 95.347 141 O VAL 65 26.155 78.411 95.384 142 CB VAL 65 28.202 81.234 94.392 143 CG1 VAL 65 29.120 81.632 93.244 144 CG2 VAL 65 27.299 82.390 94.812 145 N TRP 66 28.057 79.300 96.232 146 CA TRP 66 28.101 78.707 97.593 147 C TRP 66 27.875 77.190 97.672 148 O TRP 66 27.196 76.583 96.833 149 CB TRP 66 27.194 79.478 98.559 150 CG TRP 66 25.726 79.102 98.652 151 CD1 TRP 66 24.745 79.281 97.705 152 CD2 TRP 66 25.080 78.508 99.797 153 NE1 TRP 66 23.575 78.814 98.201 154 CE2 TRP 66 23.727 78.350 99.453 155 CE3 TRP 66 25.534 78.120 101.049 156 CZ2 TRP 66 22.841 77.803 100.369 157 CZ3 TRP 66 24.641 77.574 101.961 158 CH2 TRP 66 23.301 77.415 101.622 159 N ALA 67 28.592 76.598 98.622 160 CA ALA 67 28.546 75.152 98.917 161 C ALA 67 29.112 74.298 97.784 162 O ALA 67 28.542 74.257 96.691 163 CB ALA 67 27.114 74.727 99.232 164 N ALA 68 30.272 73.704 98.025 165 CA ALA 68 30.899 72.810 97.040 166 C ALA 68 30.854 71.336 97.455 167 O ALA 68 31.385 70.472 96.749 168 CB ALA 68 32.349 73.234 96.835 169 N TRP 69 30.228 71.063 98.589 170 CA TRP 69 30.251 69.713 99.175 171 C TRP 69 28.931 68.950 99.044 172 O TRP 69 28.827 67.808 99.507 173 CB TRP 69 30.622 69.831 100.649 174 CG TRP 69 32.028 70.344 100.891 175 CD1 TRP 69 32.391 71.587 101.364 176 CD2 TRP 69 33.255 69.613 100.675 177 NE1 TRP 69 33.746 71.641 101.434 178 CE2 TRP 69 34.301 70.482 101.031 179 CE3 TRP 69 33.532 68.333 100.220 180 CZ2 TRP 69 35.617 70.055 100.924 181 CZ3 TRP 69 34.853 67.914 100.115 182 CH2 TRP 69 35.890 68.771 100.466 183 N GLY 70 27.939 69.576 98.439 184 CA GLY 70 26.621 68.949 98.250 185 C GLY 70 26.120 69.309 96.856 186 O GLY 70 26.280 68.535 95.906 187 N PRO 71 25.472 70.459 96.756 188 CA PRO 71 25.644 71.270 95.554 189 C PRO 71 27.115 71.651 95.442 190 O PRO 71 27.831 71.677 96.455 191 CB PRO 71 24.784 72.477 95.766 192 CG PRO 71 24.328 72.502 97.218 193 CD PRO 71 24.910 71.250 97.857 194 N SER 72 27.585 71.833 94.223 195 CA SER 72 28.979 72.242 94.041 196 C SER 72 29.222 73.082 92.786 197 O SER 72 29.575 72.521 91.742 198 CB SER 72 29.839 70.987 93.955 199 OG SER 72 31.195 71.405 93.870 200 N PRO 73 29.043 74.394 92.862 201 CA PRO 73 28.155 75.062 93.815 202 C PRO 73 26.721 75.068 93.286 203 O PRO 73 26.273 74.084 92.680 204 CB PRO 73 28.687 76.457 93.849 205 CG PRO 73 29.434 76.702 92.544 206 CD PRO 73 29.478 75.351 91.846 207 N GLU 74 26.002 76.113 93.672 208 CA GLU 74 24.716 76.571 93.102 209 C GLU 74 23.547 76.238 94.024 210 O GLU 74 23.189 75.072 94.242 211 CB GLU 74 24.471 76.157 91.637 212 CG GLU 74 23.762 74.820 91.404 213 CD GLU 74 23.683 74.531 89.909 214 OE1 GLU 74 22.924 75.226 89.247 215 OE2 GLU 74 24.470 73.724 89.435 216 N GLY 75 23.015 77.287 94.624 217 CA GLY 75 21.805 77.152 95.435 218 C GLY 75 21.038 78.465 95.592 219 O GLY 75 21.235 79.192 96.575 220 N PRO 76 20.081 78.693 94.706 221 CA PRO 76 18.984 79.605 95.038 222 C PRO 76 18.225 79.017 96.220 223 O PRO 76 17.925 77.818 96.191 224 CB PRO 76 18.120 79.629 93.816 225 CG PRO 76 18.566 78.524 92.871 226 CD PRO 76 19.796 77.900 93.507 227 N PRO 77 18.010 79.819 97.256 228 CA PRO 77 17.607 79.320 98.584 229 C PRO 77 16.356 78.446 98.550 230 O PRO 77 15.217 78.914 98.455 231 CB PRO 77 17.423 80.543 99.427 232 CG PRO 77 17.944 81.751 98.666 233 CD PRO 77 18.371 81.235 97.302 234 N GLU 78 16.610 77.162 98.717 235 CA GLU 78 15.591 76.130 98.544 236 C GLU 78 14.723 76.006 99.783 237 O GLU 78 15.224 76.127 100.905 238 CB GLU 78 16.283 74.798 98.249 239 CG GLU 78 17.116 74.839 96.966 240 CD GLU 78 18.620 74.833 97.261 241 OE1 GLU 78 19.072 75.728 97.966 242 OE2 GLU 78 19.268 73.877 96.860 243 N THR 79 13.447 75.736 99.537 244 CA THR 79 12.384 75.546 100.552 245 C THR 79 12.246 76.710 101.538 246 O THR 79 13.203 77.129 102.199 247 CB THR 79 12.558 74.220 101.299 248 OG1 THR 79 13.660 74.285 102.194 249 CG2 THR 79 12.756 73.045 100.347 250 N PRO 80 11.016 77.186 101.652 251 CA PRO 80 10.706 78.342 102.501 252 C PRO 80 11.125 78.127 103.955 253 O PRO 80 10.796 77.112 104.579 254 CB PRO 80 9.225 78.540 102.376 255 CG PRO 80 8.662 77.519 101.400 256 CD PRO 80 9.841 76.680 100.939 257 N GLY 82 11.864 79.097 104.466 258 CA GLY 82 12.371 79.040 105.841 259 C GLY 82 13.088 80.333 106.220 260 O GLY 82 12.892 81.376 105.585 261 N ALA 83 14.052 80.213 107.121 262 CA ALA 83 14.754 81.394 107.657 263 C ALA 83 15.807 81.992 106.718 264 O ALA 83 16.106 83.190 106.816 265 CB ALA 83 15.409 81.012 108.979 266 N ALA 84 16.142 81.264 105.663 267 CA ALA 84 17.074 81.774 104.651 268 C ALA 84 16.392 82.738 103.679 269 O ALA 84 17.069 83.573 103.067 270 CB ALA 84 17.651 80.594 103.877 271 N LEU 85 15.073 82.819 103.774 272 CA LEU 85 14.294 83.709 102.916 273 C LEU 85 14.422 85.158 103.386 274 O LEU 85 14.589 86.050 102.545 275 CB LEU 85 12.829 83.270 102.976 276 CG LEU 85 12.387 82.353 101.829 277 CD1 LEU 85 13.232 81.093 101.654 278 CD2 LEU 85 10.912 81.996 101.968 279 N ALA 86 14.701 85.333 104.670 280 CA ALA 86 14.904 86.682 105.207 281 C ALA 86 16.311 87.185 104.900 282 O ALA 86 16.482 88.367 104.579 283 CB ALA 86 14.690 86.646 106.716 284 N GLU 87 17.217 86.248 104.667 285 CA GLU 87 18.590 86.610 104.314 286 C GLU 87 18.691 86.919 102.825 287 O GLU 87 19.450 87.813 102.433 288 CB GLU 87 19.515 85.452 104.669 289 CG GLU 87 19.423 85.092 106.149 290 CD GLU 87 19.829 86.276 107.026 291 OE1 GLU 87 18.932 86.897 107.580 292 OE2 GLU 87 21.021 86.435 107.246 293 N ALA 88 17.763 86.376 102.055 294 CA ALA 88 17.698 86.718 100.638 295 C ALA 88 17.014 88.069 100.440 296 O ALA 88 17.472 88.851 99.598 297 CB ALA 88 16.958 85.613 99.895 298 N VAL 89 16.159 88.455 101.378 299 CA VAL 89 15.590 89.809 101.342 300 C VAL 89 16.631 90.833 101.788 301 O VAL 89 16.763 91.883 101.145 302 CB VAL 89 14.380 89.901 102.266 303 CG1 VAL 89 13.820 91.317 102.270 304 CG2 VAL 89 13.291 88.918 101.864 305 N ARG 90 17.516 90.412 102.681 306 CA ARG 90 18.656 91.237 103.095 307 C ARG 90 19.509 91.650 101.901 308 O ARG 90 19.543 92.846 101.566 309 CB ARG 90 19.512 90.439 104.073 310 CG ARG 90 18.842 90.287 105.432 311 CD ARG 90 18.838 91.604 106.200 312 NE ARG 90 20.209 92.011 106.550 313 CZ ARG 90 20.804 93.099 106.058 314 NH1 ARG 90 20.177 93.848 105.151 315 NH2 ARG 90 22.050 93.404 106.428 316 N ARG 91 19.878 90.662 101.102 317 CA ARG 91 20.731 90.897 99.933 318 C ARG 91 19.997 91.558 98.760 319 O ARG 91 20.613 92.366 98.054 320 CB ARG 91 21.280 89.538 99.513 321 CG ARG 91 22.086 88.931 100.657 322 CD ARG 91 22.598 87.530 100.350 323 NE ARG 91 21.505 86.546 100.336 324 CZ ARG 91 21.621 85.331 100.877 325 NH1 ARG 91 20.629 84.445 100.758 326 NH2 ARG 91 22.762 84.975 101.472 327 N PHE 92 18.678 91.443 98.732 328 CA PHE 92 17.865 92.056 97.674 329 C PHE 92 17.611 93.546 97.932 330 O PHE 92 17.504 94.333 96.979 331 CB PHE 92 16.537 91.302 97.662 332 CG PHE 92 15.630 91.534 96.457 333 CD1 PHE 92 15.575 90.578 95.451 334 CD2 PHE 92 14.839 92.674 96.375 335 CE1 PHE 92 14.743 90.769 94.357 336 CE2 PHE 92 14.011 92.867 95.278 337 CZ PHE 92 13.962 91.914 94.270 338 N GLN 93 17.690 93.952 99.189 339 CA GLN 93 17.550 95.372 99.516 340 C GLN 93 18.848 96.111 99.247 341 O GLN 93 18.839 97.118 98.528 342 CB GLN 93 17.225 95.505 100.991 343 CG GLN 93 15.866 94.923 101.340 344 CD GLN 93 15.922 94.603 102.821 345 OE1 GLN 93 14.940 94.710 103.567 346 NE2 GLN 93 17.118 94.219 103.233 347 N ARG 94 19.955 95.439 99.524 348 CA ARG 94 21.265 96.052 99.283 349 C ARG 94 21.615 96.029 97.793 350 O ARG 94 22.449 96.812 97.324 351 CB ARG 94 22.311 95.291 100.086 352 CG ARG 94 21.836 95.082 101.519 353 CD ARG 94 22.958 94.566 102.411 354 NE ARG 94 23.691 93.464 101.772 355 CZ ARG 94 23.723 92.217 102.246 356 NH1 ARG 94 22.935 91.867 103.264 357 NH2 ARG 94 24.470 91.297 101.636 358 N ALA 95 20.873 95.224 97.052 359 CA ALA 95 20.914 95.218 95.595 360 C ALA 95 20.370 96.501 94.984 361 O ALA 95 21.121 97.418 94.626 362 CB ALA 95 19.993 94.100 95.142 363 N ASN 96 19.052 96.603 94.985 364 CA ASN 96 18.361 97.636 94.195 365 C ASN 96 18.121 98.955 94.930 366 O ASN 96 17.040 99.540 94.794 367 CB ASN 96 17.028 97.058 93.737 368 CG ASN 96 17.268 95.754 92.982 369 OD1 ASN 96 18.153 95.666 92.124 370 ND2 ASN 96 16.528 94.731 93.371 371 N ALA 97 19.141 99.433 95.632 372 CA ALA 97 19.077 100.677 96.417 373 C ALA 97 17.845 100.729 97.312 374 O ALA 97 16.945 101.555 97.119 375 CB ALA 97 19.091 101.870 95.466 376 N LEU 98 17.762 99.760 98.205 377 CA LEU 98 16.652 99.670 99.151 378 C LEU 98 17.221 99.602 100.560 379 O LEU 98 18.285 99.008 100.778 380 CB LEU 98 15.861 98.398 98.858 381 CG LEU 98 15.354 98.342 97.423 382 CD1 LEU 98 14.924 96.934 97.037 383 CD2 LEU 98 14.240 99.353 97.183 384 N PRO 99 16.516 100.198 101.507 385 CA PRO 99 16.987 100.216 102.894 386 C PRO 99 17.169 98.804 103.445 387 O PRO 99 16.319 97.929 103.244 388 CB PRO 99 15.946 100.976 103.655 389 CG PRO 99 14.841 101.407 102.703 390 CD PRO 99 15.244 100.901 101.328 391 N ALA 100 18.385 98.546 103.895 392 CA ALA 100 18.701 97.277 104.567 393 C ALA 100 17.928 97.062 105.875 394 O ALA 100 18.386 97.469 106.948 395 CB ALA 100 20.199 97.279 104.862 396 N SER 101 16.783 96.403 105.769 397 CA SER 101 15.981 96.027 106.937 398 C SER 101 15.980 94.513 107.140 399 O SER 101 16.552 93.997 108.107 400 CB SER 101 14.552 96.504 106.700 401 OG SER 101 13.747 96.061 107.785 402 N GLY 102 15.369 93.816 106.198 403 CA GLY 102 15.267 92.357 106.246 404 C GLY 102 13.848 91.898 105.926 405 O GLY 102 13.537 90.703 106.006 406 N GLU 103 12.989 92.851 105.600 407 CA GLU 103 11.577 92.532 105.343 408 C GLU 103 11.087 93.142 104.034 409 O GLU 103 11.851 93.787 103.305 410 CB GLU 103 10.712 93.024 106.500 411 CG GLU 103 11.044 92.293 107.798 412 CD GLU 103 10.019 92.630 108.873 413 OE1 GLU 103 8.981 91.979 108.880 414 OE2 GLU 103 10.231 93.606 109.577 415 N LEU 104 9.814 92.917 103.745 416 CA LEU 104 9.184 93.433 102.517 417 C LEU 104 8.858 94.921 102.629 418 O LEU 104 7.718 95.315 102.895 419 CB LEU 104 7.901 92.649 102.264 420 CG LEU 104 8.183 91.172 102.007 421 CD1 LEU 104 6.888 90.372 101.920 422 CD2 LEU 104 9.018 90.984 100.744 423 N ASP 105 9.878 95.733 102.407 424 CA ASP 105 9.754 97.188 102.503 425 C ASP 105 8.923 97.720 101.336 426 O ASP 105 8.912 97.117 100.257 427 CB ASP 105 11.160 97.789 102.503 428 CG ASP 105 12.002 97.245 103.666 429 OD1 ASP 105 11.426 96.968 104.711 430 OD2 ASP 105 13.216 97.306 103.547 431 N ALA 106 8.326 98.888 101.509 432 CA ALA 106 7.396 99.425 100.497 433 C ALA 106 8.046 99.742 99.149 434 O ALA 106 7.539 99.293 98.114 435 CB ALA 106 6.756 100.690 101.056 436 N ALA 107 9.287 100.206 99.183 437 CA ALA 107 10.012 100.478 97.936 438 C ALA 107 10.553 99.200 97.294 439 O ALA 107 10.609 99.109 96.062 440 CB ALA 107 11.164 101.428 98.242 441 N THR 108 10.622 98.145 98.091 442 CA THR 108 11.093 96.854 97.604 443 C THR 108 9.949 96.159 96.878 444 O THR 108 10.142 95.684 95.755 445 CB THR 108 11.523 96.021 98.808 446 OG1 THR 108 12.493 96.755 99.544 447 CG2 THR 108 12.134 94.687 98.401 448 N LEU 109 8.740 96.416 97.358 449 CA LEU 109 7.530 95.869 96.736 450 C LEU 109 7.124 96.651 95.487 451 O LEU 109 6.572 96.054 94.555 452 CB LEU 109 6.400 95.932 97.757 453 CG LEU 109 6.703 95.078 98.982 454 CD1 LEU 109 5.714 95.357 100.107 455 CD2 LEU 109 6.724 93.594 98.631 456 N ALA 110 7.589 97.887 95.374 457 CA ALA 110 7.357 98.678 94.158 458 C ALA 110 8.371 98.334 93.062 459 O ALA 110 8.119 98.543 91.870 460 CB ALA 110 7.446 100.157 94.512 461 N ALA 111 9.448 97.678 93.466 462 CA ALA 111 10.412 97.084 92.532 463 C ALA 111 10.062 95.626 92.217 464 O ALA 111 10.862 94.903 91.609 465 CB ALA 111 11.806 97.172 93.145 466 N MET 112 8.890 95.207 92.666 467 CA MET 112 8.390 93.844 92.483 468 C MET 112 7.006 93.871 91.842 469 O MET 112 6.099 93.172 92.310 470 CB MET 112 8.263 93.182 93.850 471 CG MET 112 9.599 92.864 94.513 472 SD MET 112 9.446 92.431 96.262 473 CE MET 112 11.063 91.687 96.562 474 N ASN 113 6.830 94.681 90.810 475 CA ASN 113 5.507 94.816 90.179 476 C ASN 113 5.495 94.487 88.671 477 O ASN 113 4.510 93.924 88.181 478 CB ASN 113 4.977 96.238 90.395 479 CG ASN 113 4.620 96.525 91.858 480 OD1 ASN 113 4.865 97.628 92.360 481 ND2 ASN 113 4.024 95.546 92.518 482 N ARG 114 6.630 94.648 88.005 483 CA ARG 114 6.721 94.570 86.526 484 C ARG 114 7.717 93.474 86.041 485 O ARG 114 8.041 92.638 86.876 486 CB ARG 114 7.086 96.003 86.142 487 CG ARG 114 7.697 96.695 87.362 488 CD ARG 114 8.115 98.140 87.116 489 NE ARG 114 8.837 98.648 88.295 490 CZ ARG 114 10.122 99.007 88.272 491 NH1 ARG 114 10.807 98.967 87.125 492 NH2 ARG 114 10.719 99.425 89.389 493 N PRO 115 7.951 93.232 84.757 494 CA PRO 115 8.798 92.052 84.357 495 C PRO 115 10.323 92.178 84.586 496 O PRO 115 10.832 93.271 84.405 497 CB PRO 115 8.522 91.906 82.900 498 CG PRO 115 7.627 93.037 82.415 499 CD PRO 115 7.316 93.897 83.619 500 N ARG 116 11.083 91.107 84.816 501 CA ARG 116 12.551 91.286 85.082 502 C ARG 116 13.596 90.574 84.158 503 O ARG 116 13.908 91.144 83.108 504 CB ARG 116 12.831 90.983 86.550 505 CG ARG 116 14.181 91.501 87.058 506 CD ARG 116 14.352 91.299 88.560 507 NE ARG 116 13.879 92.466 89.318 508 CZ ARG 116 14.379 92.814 90.506 509 NH1 ARG 116 15.319 92.066 91.075 510 NH2 ARG 116 13.930 93.908 91.124 511 N CYS 117 14.164 89.422 84.548 512 CA CYS 117 15.464 88.957 83.959 513 C CYS 117 15.915 87.456 84.062 514 O CYS 117 15.647 86.864 85.095 515 CB CYS 117 16.506 89.743 84.720 516 SG CYS 117 16.666 91.532 84.541 517 N GLY 118 16.962 87.102 83.310 518 CA GLY 118 17.597 85.743 83.076 519 C GLY 118 17.889 84.616 84.118 520 O GLY 118 16.937 84.196 84.773 521 N VAL 119 19.157 84.163 84.242 522 CA VAL 119 19.564 82.851 84.891 523 C VAL 119 20.171 82.707 86.357 524 O VAL 119 19.621 81.819 87.021 525 CB VAL 119 20.381 82.067 83.849 526 CG1 VAL 119 21.399 82.913 83.095 527 CG2 VAL 119 21.010 80.794 84.408 528 N PRO 120 21.295 83.269 86.843 529 CA PRO 120 21.590 83.253 88.321 530 C PRO 120 21.287 84.522 89.217 531 O PRO 120 22.151 85.397 89.288 532 CB PRO 120 23.072 83.064 88.306 533 CG PRO 120 23.609 83.655 87.003 534 CD PRO 120 22.385 83.940 86.142

TABLE V Atom Atom Residue No Name Residue No X coord Y coord Z coord 1 N GLY 161 30.189 93.412 91.740 2 CA GLY 161 29.826 93.284 90.328 3 C GLY 161 30.977 92.572 89.640 4 O GLY 161 31.105 91.343 89.681 5 N TYR 162 31.837 93.390 89.063 6 CA TYR 162 33.177 92.966 88.660 7 C TYR 162 33.868 92.360 89.888 8 O TYR 162 33.608 92.838 90.998 9 CB TYR 162 33.908 94.222 88.162 10 CG TYR 162 33.675 95.479 89.009 11 CD1 TYR 162 34.400 95.682 90.179 12 CD2 TYR 162 32.750 96.435 88.598 13 CE1 TYR 162 34.168 96.806 90.959 14 CE2 TYR 162 32.513 97.559 89.379 15 CZ TYR 162 33.215 97.736 90.562 16 OH TYR 162 32.888 98.775 91.403 17 N PRO 163 34.742 91.374 89.733 18 CA PRO 163 35.388 91.003 88.459 19 C PRO 163 34.563 90.120 87.512 20 O PRO 163 33.892 90.625 86.608 21 CB PRO 163 36.634 90.276 88.868 22 CG PRO 163 36.611 90.016 90.365 23 CD PRO 163 35.350 90.687 90.876 24 N ASP 164 34.791 88.817 87.608 25 CA ASP 164 34.209 87.847 86.664 26 C ASP 164 33.087 87.037 87.306 27 O ASP 164 32.958 87.000 88.535 28 CB ASP 164 35.309 86.898 86.202 29 CG ASP 164 36.490 87.679 85.634 30 OD1 ASP 164 37.586 87.461 86.127 31 OD2 ASP 164 36.247 88.610 84.878 32 N GLY 165 32.290 86.392 86.467 33 CA GLY 165 31.139 85.615 86.946 34 C GLY 165 31.536 84.305 87.615 35 O GLY 165 32.715 84.070 87.916 36 N GLY 166 30.530 83.476 87.855 37 CA GLY 166 30.719 82.161 88.491 38 C GLY 166 31.669 81.282 87.687 39 O GLY 166 31.394 80.946 86.529 40 N ALA 167 32.845 81.077 88.268 41 CA ALA 167 33.960 80.339 87.649 42 C ALA 167 34.371 80.897 86.283 43 O ALA 167 34.852 80.135 85.437 44 CB ALA 167 33.576 78.869 87.513 45 N ALA 168 34.296 82.218 86.153 46 CA ALA 168 34.545 82.966 84.906 47 C ALA 168 34.344 82.172 83.616 48 O ALA 168 35.332 81.827 82.955 49 CB ALA 168 35.973 83.498 84.944 50 N GLN 169 33.098 81.920 83.242 51 CA GLN 169 32.845 81.192 81.987 52 C GLN 169 32.920 82.109 80.767 53 O GLN 169 31.909 82.654 80.304 54 CB GLN 169 31.479 80.517 82.012 55 CG GLN 169 31.296 79.674 80.751 56 CD GLN 169 29.891 79.089 80.662 57 OE1 GLN 169 29.401 78.463 81.611 58 NE2 GLN 169 29.267 79.301 79.517 59 N ALA 170 34.138 82.313 80.305 60 CA ALA 170 34.385 83.041 79.064 61 C ALA 170 34.375 82.069 77.894 62 O ALA 170 34.285 80.847 78.077 63 CB ALA 170 35.746 83.722 79.160 64 N PHE 171 34.360 82.620 76.698 65 CA PHE 171 34.476 81.787 75.502 66 C PHE 171 35.924 81.768 75.042 67 O PHE 171 36.713 82.642 75.411 68 CB PHE 171 33.556 82.294 74.400 69 CG PHE 171 32.221 81.552 74.335 70 CD1 PHE 171 31.839 80.937 73.150 71 CD2 PHE 171 31.402 81.467 75.454 72 CE1 PHE 171 30.634 80.249 73.079 73 CE2 PHE 171 30.199 80.778 75.385 74 CZ PHE 171 29.813 80.171 74.197 75 N SER 172 36.280 80.719 74.322 76 CA SER 172 37.648 80.592 73.806 77 C SER 172 37.782 81.176 72.401 78 O SER 172 38.892 81.304 71.875 79 CB SER 172 38.021 79.117 73.782 80 OG SER 172 37.930 78.630 75.115 81 N LYS 173 36.655 81.519 71.799 82 CA LYS 173 36.682 82.134 70.473 83 C LYS 173 36.385 83.624 70.549 84 O LYS 173 35.400 84.052 71.161 85 CB LYS 173 35.652 81.449 69.580 86 CG LYS 173 36.005 79.986 69.344 87 CD LYS 173 37.352 79.852 68.639 88 CE LYS 173 37.736 78.390 68.448 89 NZ LYS 173 39.045 78.275 67.784 90 N ARG 174 37.190 84.385 69.826 91 CA ARG 174 36.998 85.841 69.715 92 C ARG 174 36.092 86.204 68.539 93 O ARG 174 35.944 87.385 68.205 94 CB ARG 174 38.352 86.514 69.499 95 CG ARG 174 39.323 86.236 70.639 96 CD ARG 174 38.800 86.761 71.974 97 NE ARG 174 39.722 86.414 73.066 98 CZ ARG 174 39.620 85.293 73.786 99 NH1 ARG 174 38.609 84.450 73.570 100 NH2 ARG 174 40.500 85.042 74.757 101 N THR 175 35.586 85.190 67.857 102 CA THR 175 34.728 85.384 66.689 103 C THR 175 33.599 84.356 66.712 104 O THR 175 33.847 83.154 66.557 105 CB THR 175 35.606 85.205 65.456 106 OG1 THR 175 36.574 86.247 65.464 107 CG2 THR 175 34.824 85.312 64.155 108 N LEU 176 32.386 84.820 66.963 109 CA LEU 176 31.251 83.894 67.120 110 C LEU 176 30.172 84.091 66.056 111 O LEU 176 29.608 85.181 65.904 112 CB LEU 176 30.639 84.069 68.505 113 CG LEU 176 31.588 83.635 69.617 114 CD1 LEU 176 30.992 83.940 70.988 115 CD2 LEU 176 31.927 82.153 69.500 116 N SER 177 29.814 82.987 65.424 117 CA SER 177 28.822 83.002 64.343 118 C SER 177 27.381 83.050 64.842 119 O SER 177 26.949 82.222 65.660 120 CB SER 177 29.015 81.760 63.483 121 OG SER 177 27.995 81.765 62.491 122 N TRP 178 26.642 83.978 64.255 123 CA TRP 178 25.217 84.198 64.524 124 C TRP 178 24.381 83.452 63.475 125 O TRP 178 24.921 83.051 62.438 126 CB TRP 178 24.990 85.718 64.453 127 CG TRP 178 23.634 86.292 64.860 128 CD1 TRP 178 22.660 85.687 65.621 129 CD2 TRP 178 23.124 87.603 64.532 130 NE1 TRP 178 21.610 86.534 65.748 131 CE2 TRP 178 21.845 87.691 65.109 132 CE3 TRP 178 23.641 88.671 63.813 133 CZ2 TRP 178 21.089 88.841 64.932 134 CZ3 TRP 178 22.879 89.822 63.650 135 CH2 TRP 178 21.607 89.905 64.207 136 N ARG 179 23.203 83.021 63.885 137 CA ARG 179 22.162 82.516 62.986 138 C ARG 179 20.915 83.356 63.231 139 O ARG 179 20.161 83.109 64.184 140 CB ARG 179 21.832 81.062 63.283 141 CG ARG 179 20.694 80.629 62.365 142 CD ARG 179 20.108 79.273 62.721 143 NE ARG 179 18.982 78.987 61.819 144 CZ ARG 179 18.500 77.766 61.585 145 NH1 ARG 179 19.002 76.714 62.231 146 NH2 ARG 179 17.489 77.603 60.729 147 N LEU 180 20.683 84.316 62.354 148 CA LEU 180 19.671 85.335 62.649 149 C LEU 180 18.229 84.890 62.341 150 O LEU 180 17.638 84.131 63.123 151 CB LEU 180 20.050 86.732 62.087 152 CG LEU 180 20.252 86.984 60.577 153 CD1 LEU 180 20.153 88.475 60.270 154 CD2 LEU 180 21.534 86.419 59.987 155 N LEU 181 17.737 85.207 61.156 156 CA LEU 181 16.307 85.112 60.840 157 C LEU 181 15.840 83.722 60.425 158 O LEU 181 14.656 83.541 60.118 159 CB LEU 181 15.975 86.118 59.742 160 CG LEU 181 15.364 87.408 60.293 161 CD1 LEU 181 14.109 87.099 61.095 162 CD2 LEU 181 16.333 88.247 61.125 163 N GLY 182 16.732 82.747 60.479 164 CA GLY 182 16.349 81.366 60.213 165 C GLY 182 15.541 80.798 61.376 166 O GLY 182 14.644 79.978 61.154 167 N GLU 183 15.816 81.255 62.590 168 CA GLU 183 15.088 80.701 63.731 169 C GLU 183 13.827 81.492 64.083 170 O GLU 183 12.783 80.863 64.299 171 CB GLU 183 16.011 80.645 64.939 172 CG GLU 183 17.222 79.742 64.766 173 CD GLU 183 16.853 78.260 64.834 174 OE1 GLU 183 16.058 77.822 64.020 175 OE2 GLU 183 17.529 77.568 65.581 176 N ALA 184 13.891 82.816 64.126 177 CA ALA 184 12.679 83.568 64.497 178 C ALA 184 12.677 85.078 64.249 179 O ALA 184 13.693 85.783 64.322 180 CB ALA 184 12.391 83.339 65.972 181 N LEU 185 11.459 85.554 64.047 182 CA LEU 185 11.152 86.984 63.914 183 C LEU 185 10.005 87.351 64.862 184 O LEU 185 9.024 86.602 64.964 185 CB LEU 185 10.723 87.233 62.467 186 CG LEU 185 10.359 88.689 62.174 187 CD1 LEU 185 11.542 89.619 62.401 188 CD2 LEU 185 9.836 88.844 60.752 189 N SER 186 10.168 88.432 65.608 190 CA SER 186 9.072 88.956 66.429 191 C SER 186 7.988 89.522 65.530 192 O SER 186 8.222 90.527 64.850 193 CB SER 186 9.584 90.084 67.313 194 OG SER 186 8.447 90.667 67.941 195 N SER 187 6.765 89.079 65.772 196 CA SER 187 5.625 89.406 64.898 197 C SER 187 5.096 90.844 64.988 198 O SER 187 4.131 91.171 64.289 199 CB SER 187 4.481 88.461 65.244 200 OG SER 187 4.077 88.745 66.577 201 N GLN 188 5.672 91.686 65.833 202 CA GLN 188 5.230 93.079 65.870 203 C GLN 188 6.164 94.015 65.104 204 O GLN 188 5.799 95.173 64.863 205 CB GLN 188 5.122 93.542 67.313 206 CG GLN 188 4.070 92.739 68.064 207 CD GLN 188 3.765 93.431 69.383 208 OE1 GLN 188 3.320 92.794 70.348 209 NE2 GLN 188 4.069 94.717 69.424 210 N LEU 189 7.327 93.529 64.708 211 CA LEU 189 8.278 94.390 63.995 212 C LEU 189 8.751 93.780 62.684 213 O LEU 189 8.687 92.566 62.469 214 CB LEU 189 9.484 94.683 64.880 215 CG LEU 189 9.141 95.714 65.947 216 CD1 LEU 189 10.361 96.046 66.793 217 CD2 LEU 189 8.595 96.977 65.294 218 N SER 190 9.234 94.648 61.813 219 CA SER 190 9.803 94.189 60.548 220 C SER 190 11.118 93.473 60.816 221 O SER 190 11.808 93.753 61.807 222 CB SER 190 10.089 95.371 59.630 223 OG SER 190 11.304 95.969 60.065 224 N VAL 191 11.542 92.712 59.824 225 CA VAL 191 12.812 91.991 59.914 226 C VAL 191 14.015 92.938 59.856 227 O VAL 191 14.974 92.729 60.608 228 CB VAL 191 12.846 90.963 58.784 229 CG1 VAL 191 12.396 91.545 57.447 230 CG2 VAL 191 14.205 90.295 58.662 231 N ALA 192 13.824 94.106 59.258 232 CA ALA 192 14.866 95.133 59.257 233 C ALA 192 15.029 95.761 60.639 234 O ALA 192 16.162 95.816 61.133 235 CB ALA 192 14.493 96.208 58.245 236 N ASP 193 13.924 95.994 61.337 237 CA ASP 193 13.993 96.527 62.709 238 C ASP 193 14.726 95.572 63.639 239 O ASP 193 15.752 95.954 64.214 240 CB ASP 193 12.590 96.703 63.290 241 CG ASP 193 11.721 97.657 62.480 242 OD1 ASP 193 12.270 98.521 61.811 243 OD2 ASP 193 10.524 97.399 62.425 244 N GLN 194 14.371 94.299 63.574 245 CA GLN 194 14.982 93.315 64.472 246 C GLN 194 16.450 93.065 64.133 247 O GLN 194 17.290 93.049 65.046 248 CB GLN 194 14.192 92.020 64.348 249 CG GLN 194 14.748 90.934 65.261 250 CD GLN 194 13.896 89.677 65.137 251 OE1 GLN 194 12.702 89.690 65.459 252 NE2 GLN 194 14.511 88.615 64.651 253 N ARG 195 16.784 93.175 62.858 254 CA ARG 195 18.164 92.990 62.421 255 C ARG 195 19.060 94.133 62.885 256 O ARG 195 20.060 93.861 63.559 257 CB ARG 195 18.178 92.949 60.900 258 CG ARG 195 19.578 92.695 60.354 259 CD ARG 195 19.612 92.878 58.843 260 NE ARG 195 18.612 92.026 58.178 261 CZ ARG 195 17.701 92.503 57.327 262 NH1 ARG 195 17.637 93.813 57.083 263 NH2 ARG 195 16.834 91.675 56.743 264 N ARG 196 18.579 95.364 62.787 265 CA ARG 196 19.430 96.504 63.147 266 C ARG 196 19.501 96.737 64.658 267 O ARG 196 20.558 97.160 65.147 268 CB ARG 196 18.935 97.758 62.422 269 CG ARG 196 17.545 98.204 62.869 270 CD ARG 196 17.018 99.356 62.024 271 NE ARG 196 16.824 98.931 60.628 272 CZ ARG 196 17.271 99.624 59.580 273 NH1 ARG 196 17.943 100.762 59.768 274 NH2 ARG 196 17.056 99.171 58.342 275 N ILE 197 18.542 96.206 65.401 276 CA ILE 197 18.596 96.331 66.856 277 C ILE 197 19.599 95.347 67.448 278 O ILE 197 20.464 95.759 68.234 279 CB ILE 197 17.207 96.061 67.420 280 CG1 ILE 197 16.230 97.149 66.991 281 CG2 ILE 197 17.259 95.962 68.938 282 CD1 ILE 197 14.804 96.824 67.421 283 N VAL 198 19.667 94.157 66.873 284 CA VAL 198 20.613 93.167 67.386 285 C VAL 198 21.999 93.363 66.760 286 O VAL 198 23.014 93.060 67.401 287 CB VAL 198 20.050 91.775 67.119 288 CG1 VAL 198 20.838 90.724 67.887 289 CG2 VAL 198 18.590 91.701 67.548 290 N ALA 199 22.038 94.118 65.674 291 CA ALA 199 23.307 94.533 65.074 292 C ALA 199 24.028 95.519 65.977 293 O ALA 199 25.164 95.250 66.392 294 CB ALA 199 23.004 95.236 63.759 295 N LEU 200 23.280 96.487 66.488 296 CA LEU 200 23.863 97.483 67.394 297 C LEU 200 24.156 96.891 68.766 298 O LEU 200 25.183 97.231 69.366 299 CB LEU 200 22.886 98.642 67.543 300 CG LEU 200 22.700 99.384 66.224 301 CD1 LEU 200 21.592 100.426 66.337 302 CD2 LEU 200 24.006 100.027 65.766 303 N ALA 201 23.401 95.865 69.131 304 CA ALA 201 23.661 95.111 70.358 305 C ALA 201 25.060 94.509 70.358 306 O ALA 201 25.902 94.928 71.164 307 CB ALA 201 22.651 93.979 70.440 308 N PHE 202 25.369 93.745 69.321 309 CA PHE 202 26.686 93.110 69.254 310 C PHE 202 27.792 94.115 68.973 311 O PHE 202 28.834 94.036 69.633 312 CB PHE 202 26.716 92.068 68.145 313 CG PHE 202 25.934 90.781 68.373 314 CD1 PHE 202 26.512 89.739 69.084 315 CD2 PHE 202 24.664 90.631 67.835 316 CE1 PHE 202 25.806 88.562 69.289 317 CE2 PHE 202 23.957 89.455 68.040 318 CZ PHE 202 24.526 88.423 68.772 319 N ARG 203 27.465 95.183 68.267 320 CA ARG 203 28.458 96.198 67.906 321 C ARG 203 28.968 97.009 69.100 322 O ARG 203 30.172 97.289 69.152 323 CB ARG 203 27.805 97.140 66.902 324 CG ARG 203 28.763 98.239 66.468 325 CD ARG 203 28.121 99.171 65.451 326 NE ARG 203 29.085 100.196 65.023 327 CZ ARG 203 29.643 100.217 63.810 328 NH1 ARG 203 29.284 99.316 62.892 329 NH2 ARG 203 30.527 101.168 63.498 330 N MET 204 28.165 97.140 70.146 331 CA MET 204 28.635 97.864 71.332 332 C MET 204 29.677 97.055 72.096 333 O MET 204 30.795 97.545 72.311 334 CB MET 204 27.451 98.143 72.252 335 CG MET 204 26.410 99.027 71.579 336 SD MET 204 26.981 100.658 71.048 337 CE MET 204 25.455 101.241 70.272 338 N TRP 205 29.438 95.756 72.195 339 CA TRP 205 30.369 94.890 72.927 340 C TRP 205 31.617 94.608 72.105 341 O TRP 205 32.726 94.771 72.633 342 CB TRP 205 29.660 93.586 73.277 343 CG TRP 205 28.486 93.808 74.202 344 CD1 TRP 205 27.149 93.766 73.877 345 CD2 TRP 205 28.551 94.108 75.613 346 NE1 TRP 205 26.421 94.065 74.985 347 CE2 TRP 205 27.231 94.277 76.040 348 CE3 TRP 205 29.609 94.271 76.498 349 CZ2 TRP 205 26.973 94.628 77.361 350 CZ3 TRP 205 29.344 94.615 77.819 351 CH2 TRP 205 28.034 94.793 78.246 352 N SER 206 31.425 94.627 70.796 353 CA SER 206 32.505 94.417 69.834 354 C SER 206 33.413 95.630 69.663 355 O SER 206 34.480 95.482 69.068 356 CB SER 206 31.876 94.129 68.474 357 OG SER 206 31.135 92.922 68.565 358 N GLU 207 33.026 96.787 70.181 359 CA GLU 207 33.855 97.988 70.054 360 C GLU 207 34.563 98.330 71.364 361 O GLU 207 35.544 99.086 71.364 362 CB GLU 207 32.935 99.141 69.653 363 CG GLU 207 33.673 100.462 69.436 364 CD GLU 207 34.511 100.435 68.158 365 OE1 GLU 207 34.203 99.591 97.327 366 OE2 GLU 207 35.120 101.465 67.893 367 N VAL 208 34.080 97.790 72.469 368 CA VAL 208 34.706 98.128 73.750 369 C VAL 208 35.660 97.023 74.200 370 O VAL 208 36.627 97.277 74.935 371 CB VAL 208 33.612 98.418 74.779 372 CG1 VAL 208 32.746 99.580 74.302 373 CG2 VAL 208 32.731 97.213 75.092 374 N THR 209 35.442 95.839 73.662 375 CA THR 209 36.304 94.685 73.906 376 C THR 209 36.395 93.935 72.579 377 O THR 209 35.384 93.867 71.871 378 CB THR 209 35.652 93.861 75.019 379 OG1 THR 209 35.767 94.615 76.221 380 CG2 THR 209 36.331 92.524 75.268 381 N PRO 210 37.594 93.520 72.189 382 CA PRO 210 37.835 93.002 70.829 383 C PRO 210 37.137 91.667 70.562 384 O PRO 210 37.676 90.593 70.859 385 CB PRO 210 39.322 92.854 70.727 386 CG PRO 210 39.964 93.206 72.060 387 CD PRO 210 38.829 93.645 72.968 388 N LEU 211 35.942 91.756 70.001 389 CA LEU 211 35.105 90.580 69.733 390 C LEU 211 34.385 90.712 68.394 391 O LEU 211 33.552 91.603 68.210 392 CB LEU 211 34.058 90.487 70.839 393 CG LEU 211 34.654 90.146 72.201 394 CD1 LEU 211 33.666 90.439 73.322 395 CD2 LEU 211 35.135 88.699 72.245 396 N ASP 212 34.637 89.782 67.495 397 CA ASP 212 34.007 89.835 66.171 398 C ASP 212 32.690 89.051 66.170 399 O ASP 212 32.669 87.827 66.362 400 CB ASP 212 35.002 89.261 65.170 401 CG ASP 212 34.553 89.522 63.737 402 OD1 ASP 212 33.768 90.439 63.539 403 OD2 ASP 212 35.007 88.796 62.866 404 N PHE 213 31.597 89.773 65.962 405 CA PHE 213 30.254 89.171 66.035 406 C PHE 213 29.314 89.556 64.887 407 O PHE 213 29.659 89.534 63.694 408 CB PHE 213 29.574 89.656 67.313 409 CG PHE 213 30.151 89.196 68.649 410 CD1 PHE 213 30.616 87.898 68.812 411 CD2 PHE 213 30.170 90.080 69.718 412 CE1 PHE 213 31.125 87.494 70.038 413 CE2 PHE 213 30.676 89.676 70.947 414 CZ PHE 213 31.156 88.381 71.105 415 N ARG 214 28.150 90.022 65.335 416 CA ARG 214 26.962 90.419 64.534 417 C ARG 214 26.908 89.948 63.085 418 O ARG 214 27.195 88.783 62.789 419 CB ARG 214 26.733 91.933 64.613 420 CG ARG 214 27.962 92.766 64.978 421 CD ARG 214 28.933 93.003 63.825 422 NE ARG 214 30.060 93.821 64.295 423 CZ ARG 214 30.118 95.143 64.118 424 NH1 ARG 214 29.157 95.768 63.436 425 NH2 ARG 214 31.159 95.834 64.590 426 N GLU 215 26.599 90.866 62.180 427 CA GLU 215 26.351 90.498 60.775 428 C GLU 215 27.592 90.096 59.980 429 O GLU 215 27.434 89.480 58.923 430 CB GLU 215 25.655 91.637 60.039 431 CG GLU 215 24.227 91.856 60.522 432 CD GLU 215 24.132 93.233 61.162 433 OE1 GLU 215 23.194 93.953 60.848 434 OE2 GLU 215 25.067 93.572 61.877 435 N ASP 216 28.775 90.207 60.566 436 CA ASP 216 29.978 89.731 59.885 437 C ASP 216 30.122 88.221 60.079 438 O ASP 216 30.820 87.549 59.312 439 CB ASP 216 31.189 90.444 60.476 440 CG ASP 216 31.054 91.951 60.281 441 OD1 ASP 216 31.111 92.658 61.279 442 OD2 ASP 216 30.727 92.354 59.175 443 N LEU 217 29.406 87.695 61.062 444 CA LEU 217 29.376 86.254 61.309 445 C LEU 217 27.961 85.698 61.196 446 O LEU 217 27.720 84.555 61.600 447 CB LEU 217 29.895 85.949 62.714 448 CG LEU 217 31.416 85.856 62.818 449 CD1 LEU 217 31.987 85.048 61.657 450 CD2 LEU 217 32.092 87.219 62.907 451 N ALA 218 27.031 86.506 60.713 452 CA ALA 218 25.619 86.117 60.739 453 C ALA 218 25.171 85.291 59.540 454 O ALA 218 24.931 85.813 58.447 455 CB ALA 218 24.775 87.378 60.813 456 N ALA 219 24.948 84.017 59.803 457 CA ALA 219 24.378 83.112 58.813 458 C ALA 219 22.856 83.140 58.917 459 O ALA 219 22.297 83.460 59.976 460 CB ALA 219 24.901 81.706 59.090 461 N PRO 220 22.200 83.011 57.777 462 CA PRO 220 20.744 82.885 57.773 463 C PRO 220 20.299 81.625 58.515 464 O PRO 220 19.610 81.726 59.537 465 CB PRO 220 20.358 82.844 56.325 466 CG PRO 220 21.616 82.885 55.465 467 CD PRO 220 22.787 82.949 56.435 468 N GLY 221 20.785 80.482 58.052 469 CA GLY 221 20.480 79.188 58.668 470 C GLY 221 21.695 78.635 59.410 471 O GLY 221 22.637 79.383 59.698 472 N ALA 222 21.653 77.336 59.680 473 CA ALA 222 22.696 76.611 60.436 474 C ALA 222 22.820 77.100 61.879 475 O ALA 222 23.262 78.226 62.130 476 CB ALA 222 24.042 76.709 59.721 477 N ALA 223 22.465 76.228 62.811 478 CA ALA 223 22.461 76.577 64.239 479 C ALA 223 23.870 76.628 64.823 480 O ALA 223 24.423 75.613 65.260 481 CB ALA 223 21.635 75.534 64.984 482 N VAL 224 24.424 77.828 64.839 483 CA VAL 224 25.789 78.039 65.322 484 C VAL 224 25.847 78.485 66.783 485 O VAL 224 25.089 77.989 67.623 486 CB VAL 224 26.454 79.036 64.383 487 CG1 VAL 224 27.138 78.314 63.228 488 CG2 VAL 224 25.435 80.042 63.862 489 N ASP 225 26.730 79.430 67.076 490 CA ASP 225 27.020 79.769 68.476 491 C ASP 225 26.006 80.765 69.017 492 O ASP 225 25.706 80.794 70.216 493 CB ASP 225 28.390 80.442 68.542 494 CG ASP 225 29.434 79.681 67.731 495 OD1 ASP 225 29.648 78.511 68.012 496 OD2 ASP 225 30.007 80.298 66.840 497 N ILE 226 25.485 81.573 68.114 498 CA ILE 226 24.522 82.605 68.478 499 C ILE 226 23.222 82.423 67.700 500 O ILE 226 23.060 83.010 66.630 501 CB ILE 226 25.180 83.928 68.096 502 CG1 ILE 226 26.536 84.094 68.773 503 CG2 ILE 226 24.288 85.114 68.423 504 CD1 ILE 226 27.219 85.378 68.325 505 N LYS 227 22.306 81.620 68.205 506 CA LYS 227 21.042 81.399 67.479 507 C LYS 227 20.054 82.530 67.797 508 O LYS 227 20.390 83.379 68.624 509 CB LYS 227 20.517 80.040 67.906 510 CG LYS 227 21.589 78.973 67.715 511 CD LYS 227 21.190 77.666 68.388 512 CE LYS 227 19.871 77.142 67.843 513 NZ LYS 227 19.475 75.897 68.521 514 N LEU 228 18.924 82.616 67.106 515 CA LEU 228 17.958 83.708 67.380 516 C LEU 228 16.500 83.230 67.437 517 O LEU 228 15.839 83.116 66.401 518 CB LEU 228 18.169 84.780 66.303 519 CG LEU 228 17.094 85.867 66.181 520 CD1 LEU 228 16.733 86.546 67.495 521 CD2 LEU 228 17.526 86.915 65.165 522 N GLY 229 15.968 83.083 68.642 523 CA GLY 229 14.619 82.531 68.792 524 C GLY 229 13.696 83.225 69.793 525 O GLY 229 14.088 83.691 70.870 526 N PHE 230 12.434 83.242 69.400 527 CA PHE 230 11.342 83.810 70.198 528 C PHE 230 10.323 82.716 70.520 529 O PHE 230 9.864 82.003 69.622 530 CB PHE 230 10.634 84.904 69.392 531 CG PHE 230 11.408 86.198 69.121 532 CD1 PHE 230 12.359 86.259 68.111 533 CD2 PHE 230 11.130 87.332 69.875 534 CE1 PHE 230 13.054 87.438 67.877 535 CE2 PHE 230 11.824 88.511 69.641 536 CZ PHE 230 12.790 88.565 68.645 537 N GLY 231 9.968 82.605 71.785 538 CA GLY 231 8.952 81.640 72.213 539 C GLY 231 7.973 82.274 73.198 540 O GLY 231 7.897 83.504 73.335 541 N ARG 232 7.138 81.428 73.772 542 CA ARG 232 6.163 81.866 74.774 543 C ARG 232 5.984 80.791 75.833 544 O ARG 232 6.709 79.791 75.797 545 CB ARG 232 4.843 82.167 74.086 546 CG ARG 232 4.379 81.031 73.186 547 CD ARG 232 3.182 81.492 72.368 548 NE ARG 232 3.517 82.751 71.682 549 CZ ARG 232 2.801 83.871 71.809 550 NH1 ARG 232 1.670 83.864 72.519 551 NH2 ARG 232 3.193 84.987 71.191 552 N GLY 233 5.138 81.098 76.810 553 CA GLY 233 4.765 80.230 77.953 554 C GLY 233 5.433 78.858 78.057 555 O GLY 233 6.665 78.732 78.136 556 N ARG 234 4.598 77.836 78.089 557 CA ARG 234 5.071 76.451 78.176 558 C ARG 234 5.638 75.900 76.863 559 O ARG 234 4.906 75.442 75.983 560 CB ARG 234 3.926 75.577 78.702 561 CG ARG 234 2.523 76.109 78.379 562 CD ARG 234 2.038 75.833 76.953 563 NE ARG 234 1.715 77.093 76.259 564 CZ ARG 234 1.681 77.217 74.931 565 NH1 ARG 234 1.829 76.140 74.156 566 NH2 ARG 234 1.418 78.405 74.379 567 N HIS 235 6.952 75.974 76.740 568 CA HIS 235 7.630 75.333 75.610 569 C HIS 235 8.662 74.305 76.083 570 O HIS 235 9.246 73.596 75.257 571 CB HIS 235 8.246 76.378 74.680 572 CG HIS 235 9.367 77.239 75.231 573 ND1 HIS 235 9.237 78.363 75.961 574 CD2 HIS 235 10.714 77.032 75.050 575 CE1 HIS 235 10.461 78.849 76.249 576 NE2 HIS 235 11.373 78.029 75.682 577 N LEU 236 8.820 74.209 77.399 578 CA LEU 236 9.673 73.200 78.053 579 C LEU 236 11.128 73.258 77.594 580 O LEU 236 11.509 72.691 76.565 581 CB LEU 236 9.110 71.802 77.798 582 CG LEU 236 9.900 70.730 78.545 583 CD1 LEU 236 9.814 70.936 80.054 584 CD2 LEU 236 9.418 69.332 78.170 585 N GLY 237 11.951 73.863 78.428 586 CA GLY 237 13.380 73.931 78.139 587 C GLY 237 14.195 73.415 79.312 588 O GLY 237 15.299 73.909 79.578 589 N CYS 238 13.581 72.497 80.042 590 CA CYS 238 14.236 71.716 81.102 591 C CYS 238 14.798 72.524 82.271 592 O CYS 238 15.798 73.242 82.152 593 CB CYS 238 15.349 70.881 80.478 594 SG CYS 238 14.805 69.652 79.269 595 N PRO 239 14.161 72.355 83.417 596 CA PRO 239 12.826 71.750 83.500 597 C PRO 239 11.649 72.715 83.261 598 O PRO 239 10.501 72.258 83.222 599 CB PRO 239 12.766 71.261 84.915 600 CG PRO 239 13.825 71.992 85.731 601 CD PRO 239 14.651 72.783 84.727 602 N ARG 240 11.904 74.000 83.062 603 CA ARG 240 10.806 74.969 83.122 604 C ARG 240 10.493 75.647 81.787 605 O ARG 240 10.789 75.126 80.703 606 CB ARG 240 11.137 76.060 84.137 607 CG ARG 240 12.001 75.627 85.324 608 CD ARG 240 13.455 76.049 85.105 609 NE ARG 240 14.285 75.957 86.312 610 CZ ARG 240 15.439 76.621 86.424 611 NH1 ARG 240 16.230 76.412 87.477 612 NH2 ARG 240 15.847 77.414 85.432 613 N ALA 241 9.701 76.703 81.910 614 CA ALA 241 9.254 77.522 80.774 615 C ALA 241 8.860 78.937 81.233 616 O ALA 241 9.162 79.335 82.364 617 CB ALA 241 8.062 76.826 80.139 618 N PHE 242 8.229 79.689 80.345 619 CA PHE 242 7.740 81.045 80.659 620 C PHE 242 6.377 81.045 81.357 621 O PHE 242 5.616 80.072 81.315 622 CB PHE 242 7.522 81.848 79.378 623 CG PHE 242 8.716 82.366 78.589 624 CD1 PHE 242 9.500 83.394 79.094 625 CD2 PHE 242 8.980 81.854 77.328 626 CE1 PHE 242 10.569 83.884 78.359 627 CE2 PHE 242 10.047 82.343 76.588 628 CZ PHE 242 10.844 83.355 77.107 629 N ASP 243 6.051 82.195 81.931 630 CA ASP 243 4.698 82.440 82.452 631 C ASP 243 3.903 83.297 81.458 632 O ASP 243 2.672 83.364 81.538 633 CB ASP 243 4.751 83.119 83.828 634 CG ASP 243 4.871 84.642 83.736 635 OD1 ASP 243 5.923 85.104 83.320 636 OD2 ASP 243 3.864 85.304 83.937 637 N GLY 244 4.592 83.904 80.502 638 CA GLY 244 3.887 84.632 79.442 639 C GLY 244 4.052 86.153 79.441 640 O GLY 244 5.117 86.708 79.747 641 N SER 245 2.943 86.800 79.116 642 CA SER 245 2.901 88.236 78.789 643 C SER 245 3.100 89.170 79.971 644 O SER 245 2.728 88.855 81.108 645 CB SER 245 1.532 88.550 78.191 646 OG SER 245 1.355 87.764 77.021 647 N GLY 246 3.770 90.278 79.695 648 CA GLY 246 3.888 91.393 80.642 649 C GLY 246 5.050 91.244 81.617 650 O GLY 246 5.997 92.040 81.619 651 N GLN 247 4.990 90.169 82.380 652 CA GLN 247 5.900 89.945 83.493 653 C GLN 247 7.280 89.401 83.172 654 O GLN 247 7.969 89.145 84.162 655 CB GLN 247 5.258 88.995 84.500 656 CG GLN 247 4.246 89.705 85.392 657 CD GLN 247 4.908 90.884 86.111 658 OE1 GLN 247 4.744 92.037 85.695 659 NE2 GLN 247 5.658 90.589 87.159 660 N GLU 248 7.711 89.201 81.931 661 CA GLU 248 9.103 88.735 81.796 662 C GLU 248 9.851 88.817 80.466 663 O GLU 248 9.342 88.723 79.340 664 CB GLU 248 9.215 87.303 82.287 665 CG GLU 248 8.282 86.350 81.579 666 CD GLU 248 8.506 84.990 82.204 667 OE1 GLU 248 8.181 84.012 81.545 668 OE2 GLU 248 8.957 84.926 83.337 669 N PHE 249 11.124 88.611 80.760 670 CA PHE 249 12.323 88.534 79.908 671 C PHE 249 12.382 87.686 78.644 672 O PHE 249 11.414 87.372 77.943 673 CB PHE 249 13.374 87.872 80.807 674 CG PHE 249 13.045 86.515 81.478 675 CD1 PHE 249 13.800 86.126 82.579 676 CD2 PHE 249 12.065 85.652 80.998 677 CE1 PHE 249 13.525 84.932 83.237 678 CE2 PHE 249 11.786 84.463 81.656 679 CZ PHE 249 12.507 84.109 82.783 680 N ALA 250 13.650 87.586 78.277 681 CA ALA 250 14.203 86.574 77.385 682 C ALA 250 15.205 85.794 78.241 683 O ALA 250 16.146 86.379 78.792 684 CB ALA 250 14.936 87.272 76.255 685 N HIS 251 14.954 84.513 78.422 686 CA HIS 251 15.715 83.734 79.411 687 C HIS 251 16.825 82.835 78.871 688 O HIS 251 16.606 81.966 78.024 689 CB HIS 251 14.729 82.911 80.214 690 CG HIS 251 13.729 82.007 79.509 691 ND1 HIS 251 12.545 81.629 80.025 692 CD2 HIS 251 13.829 81.397 78.278 693 CE1 HIS 251 11.912 80.815 79.163 694 NE2 HIS 251 12.706 80.676 78.079 695 N ALA 252 17.998 82.977 79.453 696 CA ALA 252 19.161 82.213 78.984 697 C ALA 252 19.548 81.052 79.878 698 O ALA 252 18.698 80.337 80.427 699 CB ALA 252 20.368 83.140 78.889 700 N TRP 253 20.823 80.736 79.719 701 CA TRP 253 21.608 79.883 80.627 702 C TRP 253 23.053 79.802 80.146 703 O TRP 253 23.408 80.390 79.114 704 CB TRP 253 21.024 78.483 80.786 705 CG TRP 253 20.413 78.263 82.160 706 CD1 TRP 253 19.095 77.977 82.441 707 CD2 TRP 253 21.098 78.318 83.432 708 NE1 TRP 253 18.956 77.852 83.785 709 CE2 TRP 253 20.130 78.047 84.415 710 CE3 TRP 253 22.412 78.560 83.802 711 CZ2 TRP 253 20.494 78.013 85.753 712 CZ3 TRP 253 22.770 78.526 85.144 713 CH2 TRP 253 21.815 78.254 86.115 714 N ARG 254 23.804 78.899 80.761 715 CA ARG 254 25.247 78.736 80.508 716 C ARG 254 25.611 78.040 79.193 717 O ARG 254 26.800 77.925 78.876 718 CB ARG 254 25.870 77.974 81.669 719 CG ARG 254 25.906 78.830 82.929 720 CD ARG 254 26.750 80.083 82.717 721 NE ARG 254 26.795 80.908 83.933 722 CZ ARG 254 27.832 80.926 84.776 723 NH1 ARG 254 28.900 80.161 84.541 724 NH2 ARG 254 27.797 81.706 85.857 725 N LEU 255 24.619 77.716 78.377 726 CA LEU 255 24.887 77.247 77.015 727 C LEU 255 25.200 78.444 76.119 728 O LEU 255 25.676 78.287 74.992 729 CB LEU 255 23.678 76.500 76.445 730 CG LEU 255 23.665 75.001 76.754 731 CD1 LEU 255 23.350 74.688 78.214 732 CD2 LEU 255 22.654 74.294 75.858 733 N GLY 256 24.905 79.630 76.632 734 CA GLY 256 25.311 80.871 75.989 735 C GLY 256 24.135 81.625 75.404 736 O GLY 256 24.298 82.731 74.882 737 N ASP 257 22.958 81.027 75.476 738 CA ASP 257 21.829 81.600 74.750 739 C ASP 257 20.513 81.738 75.532 740 O ASP 257 20.193 80.901 76.389 741 CB ASP 257 21.600 80.732 73.517 742 CG ASP 257 22.672 80.867 72.429 743 OD1 ASP 257 22.339 81.398 71.372 744 OD2 ASP 257 23.700 80.232 72.590 745 N ILE 258 19.819 82.842 75.273 746 CA ILE 258 18.457 83.122 75.765 747 C ILE 258 17.406 82.537 74.852 748 O ILE 258 17.719 81.777 73.941 749 CB ILE 258 18.144 84.625 75.793 750 CG1 ILE 258 18.451 85.280 74.461 751 CG2 ILE 258 18.811 85.375 76.929 752 CD1 ILE 258 18.159 86.773 74.476 753 N HIS 259 16.176 82.667 75.298 754 CA HIS 259 15.015 82.507 74.431 755 C HIS 259 14.077 83.673 74.707 756 O HIS 259 13.759 83.935 75.874 757 CB HIS 259 14.329 81.201 74.790 758 CG HIS 259 14.111 80.276 73.619 759 ND1 HIS 259 14.319 78.949 73.614 760 CD2 HIS 259 13.681 80.620 72.359 761 CE1 HIS 259 14.021 78.454 72.396 762 NE2 HIS 259 13.626 79.490 71.621 763 N PHE 260 13.679 84.398 73.679 764 CA PHE 260 12.818 85.574 73.889 765 C PHE 260 11.382 85.221 74.243 766 O PHE 260 10.908 84.133 73.905 767 CB PHE 260 12.783 86.405 72.620 768 CG PHE 260 14.008 87.265 72.362 769 CD1 PHE 260 14.930 86.901 71.388 770 CD2 PHE 260 14.179 88.438 73.080 771 CE1 PHE 260 16.037 87.708 71.151 772 CE2 PHE 260 15.284 89.239 72.845 773 CZ PHE 260 16.213 88.876 71.882 774 N ASP 261 10.741 86.084 75.010 775 CA ASP 261 9.293 85.967 75.214 776 C ASP 261 8.599 86.908 74.237 777 O ASP 261 8.501 88.113 74.503 778 CB ASP 261 8.980 86.357 76.658 779 CG ASP 261 7.509 86.207 77.022 780 OD1 ASP 261 7.132 85.162 77.536 781 OD2 ASP 261 6.768 87.129 76.698 782 N ASP 262 7.931 86.339 73.243 783 CA ASP 262 7.285 87.134 72.179 784 C ASP 262 5.882 87.615 72.582 785 O ASP 262 5.116 88.134 71.763 786 CB ASP 262 7.219 86.304 70.897 787 CG ASP 262 7.115 87.222 69.674 788 OD1 ASP 262 7.918 88.140 69.587 789 OD2 ASP 262 6.291 86.950 68.811 790 N ASP 263 5.555 87.416 73.849 791 CA ASP 263 4.325 87.933 74.442 792 C ASP 263 4.578 89.359 74.927 793 O ASP 263 3.637 90.099 75.231 794 CB ASP 263 3.982 87.097 75.666 795 CG ASP 263 3.920 85.598 75.406 796 OD1 ASP 263 2.815 85.080 75.466 797 OD2 ASP 263 4.970 84.971 75.449 798 N GLU 264 5.851 89.695 75.076 799 CA GLU 264 6.246 91.085 75.289 800 C GLU 264 5.957 91.844 74.003 801 O GLU 264 6.333 91.394 72.914 802 CB GLU 264 7.738 91.142 75.587 803 CG GLU 264 8.135 90.410 76.863 804 CD GLU 264 7.495 91.055 78.087 805 OE1 GLU 264 7.910 92.147 78.453 806 OE2 GLU 264 6.510 90.502 78.565 807 N HIS 265 5.344 93.005 74.138 808 CA HIS 265 4.854 93.739 72.966 809 C HIS 265 5.883 94.675 72.350 810 O HIS 265 5.651 95.885 72.281 811 CB HIS 265 3.613 94.513 73.384 812 CG HIS 265 2.498 93.575 73.792 813 ND1 HIS 265 2.005 92.571 73.044 814 CD2 HIS 265 1.809 93.569 74.980 815 CE1 HIS 265 1.029 91.942 73.729 816 NE2 HIS 265 0.910 92.559 74.928 817 N PHE 266 6.821 94.056 71.652 818 CA PHE 266 7.986 94.727 71.063 819 C PHE 266 7.665 95.850 70.076 820 O PHE 266 7.202 95.635 68.948 821 CB PHE 266 8.817 93.655 70.367 822 CG PHE 266 9.360 92.593 71.318 823 CD1 PHE 266 9.154 91.246 71.052 824 CD2 PHE 266 10.057 92.976 72.458 825 CE1 PHE 266 9.648 90.282 71.920 826 CE2 PHE 266 10.554 92.012 73.324 827 CZ PHE 266 10.351 90.664 73.055 828 N THR 267 7.874 97.055 70.575 829 CA THR 267 7.864 98.292 69.801 830 C THR 267 9.307 98.607 69.458 831 O THR 267 10.217 98.070 70.101 832 CB THR 267 7.348 99.428 70.683 833 OG1 THR 267 8.335 99.712 71.673 834 CG2 THR 267 6.035 99.080 71.370 835 N PRO 268 9.529 99.366 68.402 836 CA PRO 268 10.871 99.893 68.169 837 C PRO 268 11.421 100.707 69.370 838 O PRO 268 12.151 100.078 70.149 839 CB PRO 268 10.794 100.613 66.853 840 CG PRO 268 9.355 100.567 66.349 841 CD PRO 268 8.552 99.836 67.415 842 N PRO 269 10.971 101.930 69.670 843 CA PRO 269 11.722 102.774 70.612 844 C PRO 269 11.526 102.355 72.064 845 O PRO 269 10.839 101.367 72.360 846 CB PRO 269 11.219 104.168 70.410 847 CG PRO 269 9.974 104.124 69.545 848 CD PRO 269 9.823 102.676 69.120 849 N THR 270 12.209 103.073 72.941 850 CA THR 270 12.080 102.870 74.390 851 C THR 270 10.668 103.139 74.907 852 O THR 270 10.166 104.268 74.945 853 CB THR 270 13.082 103.768 75.104 854 OG1 THR 270 14.361 103.187 74.898 855 CG2 THR 270 12.838 103.824 76.610 856 N SER 271 10.023 102.033 75.223 857 CA SER 271 8.725 102.006 75.884 858 C SER 271 8.877 101.207 77.171 859 O SER 271 9.920 100.585 77.399 860 CB SER 271 7.699 101.361 74.959 861 OG SER 271 8.160 100.058 74.630 862 N ASP 272 7.825 101.203 77.972 863 CA ASP 272 7.846 100.586 79.308 864 C ASP 272 8.206 99.097 79.254 865 O ASP 272 8.051 98.452 78.210 866 CB ASP 272 6.456 100.771 79.914 867 CG ASP 272 6.537 101.073 81.410 868 OD1 ASP 272 5.623 101.715 81.905 869 OD2 ASP 272 7.533 100.705 82.017 870 N THR 273 8.699 98.586 80.378 871 CA THR 273 9.075 97.170 80.578 872 C THR 273 9.740 96.511 79.369 873 O THR 273 10.442 97.169 78.590 874 CB THR 273 7.825 96.401 81.004 875 OG1 THR 273 6.798 96.603 80.038 876 CG2 THR 273 7.300 96.917 82.341 877 N GLY 274 9.435 95.242 79.144 878 CA GLY 274 10.019 94.515 78.003 879 C GLY 274 9.228 94.703 76.702 880 O GLY 274 9.133 93.791 75.874 881 N ILE 275 8.706 95.904 76.517 882 CA ILE 275 7.943 96.271 75.335 883 C ILE 275 8.849 97.060 74.393 884 O ILE 275 8.506 97.286 73.232 885 CB ILE 275 6.763 97.106 75.836 886 CG1 ILE 275 5.900 96.286 76.785 887 CG2 ILE 275 5.902 97.674 74.718 888 CD1 ILE 275 4.730 97.108 77.313 889 N SER 276 10.014 97.450 74.876 890 CA SER 276 10.972 98.145 73.998 891 C SER 276 12.008 97.204 73.394 892 O SER 276 13.059 96.991 74.008 893 CB SER 276 11.687 99.237 74.784 894 OG SER 276 12.153 98.729 76.028 895 N LEU 277 11.864 96.901 72.113 896 CA LEU 277 12.783 95.946 71.480 897 C LEU 277 14.123 96.597 71.136 898 O LEU 277 15.147 95.913 71.246 899 CB LEU 277 12.146 95.356 70.224 900 CG LEU 277 12.992 94.218 69.645 901 CD1 LEU 277 13.257 93.137 70.687 902 CD2 LEU 277 12.350 93.604 68.407 903 N LEU 278 14.156 97.924 71.059 904 CA LEU 278 15.437 98.621 70.867 905 C LEU 278 16.263 98.629 72.153 906 O LEU 278 17.496 98.600 72.089 907 CB LEU 278 15.172 100.060 70.443 908 CG LEU 278 15.872 100.419 69.137 909 CD1 LEU 278 15.401 101.776 68.627 910 CD2 LEU 278 17.392 100.389 69.273 911 N LYS 279 15.603 98.426 73.280 912 CA LYS 279 16.333 98.280 74.528 913 C LYS 279 16.791 96.850 74.708 914 O LYS 279 18.002 96.631 74.837 915 CB LYS 279 15.421 98.601 75.701 916 CG LYS 279 15.465 100.061 76.100 917 CD LYS 279 14.698 100.253 77.400 918 CE LYS 279 15.140 101.526 78.094 919 NZ LYS 279 16.574 101.439 78.398 920 N VAL 280 15.919 95.932 74.318 921 CA VAL 280 16.003 94.516 74.708 922 C VAL 280 17.194 93.728 74.148 923 O VAL 280 17.653 92.799 74.826 924 CB VAL 280 14.675 93.867 74.294 925 CG1 VAL 280 14.735 92.349 74.189 926 CG2 VAL 280 13.536 94.278 75.217 927 N ALA 281 17.857 94.226 73.122 928 CA ALA 281 18.981 93.462 72.592 929 C ALA 281 20.369 93.927 73.040 930 O ALA 281 21.274 93.085 73.106 931 CB ALA 281 18.909 93.495 71.072 932 N VAL 282 20.504 95.139 73.557 933 CA VAL 282 21.848 95.739 73.600 934 C VAL 282 22.752 95.352 74.785 935 O VAL 282 23.944 95.673 74.751 936 CB VAL 282 21.700 97.252 73.512 937 CG1 VAL 282 22.883 97.840 72.749 938 CG2 VAL 282 20.417 97.614 72.775 939 N HIS 283 22.263 94.596 75.752 940 CA HIS 283 23.158 94.141 76.828 941 C HIS 283 23.428 92.649 76.715 942 O HIS 283 24.424 92.171 77.281 943 CB HIS 283 22.547 94.562 78.173 944 CG HIS 283 22.351 93.608 79.356 945 ND1 HIS 283 22.098 94.019 80.613 946 CD2 HIS 283 22.345 92.228 79.401 947 CE1 HIS 283 21.962 92.959 81.423 948 NE2 HIS 283 22.111 91.847 80.670 949 N GLU 284 22.713 92.007 75.799 950 CA GLU 284 22.609 90.538 75.733 951 C GLU 284 23.910 89.776 75.906 952 O GLU 284 24.125 89.123 76.936 953 CB GLU 284 22.049 90.139 74.374 954 CG GLU 284 20.549 90.383 74.286 955 CD GLU 284 19.984 89.762 73.015 956 OE1 GLU 284 20.649 88.896 72.453 957 OE2 GLU 284 19.004 90.302 72.512 958 N ILE 285 24.854 90.114 75.050 959 CA ILE 285 26.078 89.336 74.875 960 C ILE 285 27.088 89.462 76.018 961 O ILE 285 27.896 88.541 76.194 962 CB ILE 285 26.698 89.835 73.574 963 CG1 ILE 285 25.631 89.909 72.487 964 CG2 ILE 285 27.836 88.927 73.126 965 CD1 ILE 285 25.326 91.346 72.070 966 N GLY 286 26.877 90.404 76.922 967 CA GLY 286 27.794 90.603 78.048 968 C GLY 286 27.983 89.357 78.915 969 O GLY 286 29.116 88.876 79.066 970 N HIS 287 26.893 88.727 79.319 971 CA HIS 287 27.021 87.630 80.285 972 C HIS 287 27.539 86.302 79.716 973 O HIS 287 28.266 85.626 80.452 974 CB HIS 287 25.692 87.446 81.001 975 CG HIS 287 25.371 88.628 81.892 976 ND1 HIS 287 26.050 88.997 82.994 977 CD2 HIS 287 24.353 89.537 81.730 978 CE1 HIS 287 25.491 90.105 83.518 979 NE2 HIS 287 24.440 90.437 82.736 980 N VAL 288 27.510 86.113 78.402 981 CA VAL 288 28.062 84.868 77.823 982 C VAL 288 29.572 84.982 77.617 983 O VAL 288 30.270 83.996 77.354 984 CB VAL 288 27.392 84.569 76.481 985 CG1 VAL 288 27.684 85.638 75.439 986 CG2 VAL 288 27.786 83.209 75.930 987 N LEU 289 30.082 86.182 77.837 988 CA LEU 289 31.506 86.423 77.712 989 C LEU 289 32.225 86.201 79.042 990 O LEU 289 33.460 86.222 79.085 991 CB LEU 289 31.672 87.856 77.226 992 CG LEU 289 30.950 88.065 75.896 993 CD1 LEU 289 30.907 89.539 75.509 994 CD2 LEU 289 31.552 87.222 74.775 995 N GLY 290 31.462 85.940 80.095 996 CA GLY 290 32.045 85.669 81.409 997 C GLY 290 31.651 86.726 82.430 998 O GLY 290 32.132 86.702 83.574 999 N LEU 291 30.769 87.627 82.026 1000 CA LEU 291 30.389 88.743 82.899 1001 C LEU 291 29.498 88.305 84.046 1002 O LEU 291 28.516 87.576 83.864 1003 CB LEU 291 29.672 89.828 82.101 1004 CG LEU 291 30.664 90.692 81.341 1005 CD1 LEU 291 29.963 91.853 80.647 1006 CD2 LEU 291 31.706 91.230 82.304 1007 N PRO 292 29.826 88.809 85.220 1008 CA PRO 292 28.911 88.765 86.344 1009 C PRO 292 27.913 89.894 86.212 1010 O PRO 292 27.958 90.704 85.278 1011 CB PRO 292 29.767 89.029 87.535 1012 CG PRO 292 30.999 89.763 87.039 1013 CD PRO 292 30.984 89.646 85.522 1014 N HIS 293 26.997 89.912 87.152 1015 CA HIS 293 26.067 91.019 87.259 1016 C HIS 293 26.838 92.246 87.735 1017 O HIS 293 27.814 92.093 88.475 1018 CB HIS 293 25.055 90.603 88.309 1019 CG HIS 293 23.788 91.388 88.218 1020 ND1 HIS 293 23.258 91.883 87.084 1021 CD2 HIS 293 22.957 91.758 89.245 1022 CE1 HIS 293 22.112 92.508 87.371 1023 NE2 HIS 293 21.929 92.442 88.707 1024 N THR 294 26.509 93.428 87.249 1025 CA THR 294 27.192 94.600 87.799 1026 C THR 294 26.397 95.066 89.004 1027 O THR 294 25.244 94.665 89.175 1028 CB THR 294 27.357 95.709 86.769 1029 OG1 THR 294 26.076 96.171 86.374 1030 CG2 THR 294 28.115 95.221 85.539 1031 N TYR 295 27.073 95.722 89.926 1032 CA TYR 295 26.403 96.165 91.148 1033 C TYR 295 26.607 97.668 91.375 1034 O TYR 295 26.108 98.246 92.348 1035 CB TYR 295 26.893 95.326 92.328 1036 CG TYR 295 26.365 93.886 92.476 1037 CD1 TYR 295 25.662 93.536 93.618 1038 CD2 TYR 295 26.616 92.921 91.506 1039 CE1 TYR 295 25.175 92.245 93.774 1040 CE2 TYR 295 26.135 91.626 91.661 1041 CZ TYR 295 25.407 91.293 92.794 1042 OH TYR 295 24.861 90.032 92.914 1043 N ARG 296 27.375 98.283 90.491 1044 CA ARG 296 27.478 99.749 90.450 1045 C ARG 296 26.258 100.349 89.750 1046 O ARG 296 26.140 100.212 88.525 1047 CB ARG 296 28.752 100.106 89.704 1048 CG ARG 296 29.912 100.292 90.670 1049 CD ARG 296 29.645 101.518 91.533 1050 NE ARG 296 30.715 101.758 92.509 1051 CZ ARG 296 31.583 102.767 92.432 1052 NH1 ARG 296 32.358 103.050 93.478 1053 NH2 ARG 296 31.548 103.601 91.392 1054 N THR 297 25.591 101.263 90.444 1055 CA THR 297 24.194 101.683 90.138 1056 C THR 297 23.883 102.447 88.835 1057 O THR 297 22.702 102.698 88.569 1058 CB THR 297 23.709 102.562 91.284 1059 OG1 THR 297 24.433 103.782 91.241 1060 CG2 THR 297 23.911 101.908 92.648 1061 N GLY 298 24.861 102.683 87.980 1062 CA GLY 298 24.614 103.386 86.717 1063 C GLY 298 24.901 102.480 85.526 1064 O GLY 298 24.684 102.847 84.368 1065 N SER 299 25.463 101.325 85.835 1066 CA SER 299 25.779 100.298 84.834 1067 C SER 299 24.513 99.771 84.162 1068 O SER 299 23.401 100.076 84.599 1069 CB SER 299 26.544 99.201 85.566 1070 OG SER 299 27.123 98.298 84.645 1071 N ILE 300 24.717 99.157 83.008 1072 CA ILE 300 23.662 98.624 82.130 1073 C ILE 300 23.331 97.146 82.423 1074 O ILE 300 22.449 96.594 81.756 1075 CB ILE 300 24.151 98.883 80.694 1076 CG1 ILE 300 23.971 100.372 80.401 1077 CG2 ILE 300 23.388 98.125 79.599 1078 CD1 ILE 300 22.503 100.700 80.475 1079 N MET 301 24.002 96.523 83.386 1080 CA MET 301 23.636 95.144 83.763 1081 C MET 301 23.159 95.067 85.231 1082 O MET 301 23.920 94.527 86.045 1083 CB MET 301 24.859 94.224 83.669 1084 CG MET 301 25.916 94.670 82.654 1085 SD MET 301 25.603 94.459 80.889 1086 CE MET 301 26.043 92.719 80.675 1087 N GLN 302 21.897 95.398 85.499 1088 CA GLN 302 21.335 95.540 86.864 1089 C GLN 302 19.780 95.571 86.942 1090 O GLN 302 19.092 95.665 85.923 1091 CB GLN 302 21.947 96.809 87.426 1092 CG GLN 302 23.221 96.548 88.193 1093 CD GLN 302 23.852 97.890 88.441 1094 OE1 GLN 302 24.379 98.180 89.521 1095 NE2 GLN 302 23.675 98.744 87.455 1096 N PRO 303 19.237 95.419 88.148 1097 CA PRO 303 17.795 95.154 88.360 1098 C PRO 303 16.801 96.323 88.395 1099 O PRO 303 15.689 96.100 88.886 1100 CB PRO 303 17.724 94.579 89.725 1101 CG PRO 303 18.986 94.898 90.470 1102 CD PRO 303 19.979 95.244 89.396 1103 N ASN 304 17.189 97.548 88.109 1104 CA ASN 304 16.212 98.651 88.106 1105 C ASN 304 16.073 99.308 86.727 1106 O ASN 304 16.972 99.233 85.883 1107 CB ASN 304 16.600 99.686 89.167 1108 CG ASN 304 15.871 99.509 90.509 1109 OD1 ASN 304 15.844 100.451 91.311 1110 ND2 ASN 304 15.285 98.351 90.747 1111 N TYR 305 14.914 99.902 86.494 1112 CA TYR 305 14.583 100.466 85.177 1113 C TYR 305 15.017 101.919 85.032 1114 O TYR 305 15.006 102.665 86.011 1115 CB TYR 305 13.065 100.364 85.021 1116 CG TYR 305 12.475 100.726 83.651 1117 CD1 TYR 305 11.853 101.949 83.456 1118 CD2 TYR 305 12.559 99.803 82.603 1119 CE1 TYR 305 11.318 102.239 82.208 1120 CE2 TYR 305 12.035 100.106 81.352 1121 CZ TYR 305 11.419 101.330 81.159 1122 OH TYR 305 10.862 101.620 79.936 1123 N ILE 306 15.514 102.260 83.851 1124 CA ILE 306 15.818 103.654 83.480 1125 C ILE 306 15.835 103.813 81.952 1126 O ILE 306 16.570 103.097 81.262 1127 CB ILE 306 17.155 104.062 84.100 1128 CG1 ILE 306 17.560 105.484 83.720 1129 CG2 ILE 306 18.257 103.071 83.754 1130 CD1 ILE 306 16.661 106.530 84.375 1131 N PRO 307 14.968 104.678 81.444 1132 CA PRO 307 14.881 104.923 80.002 1133 C PRO 307 16.172 105.487 79.413 1134 O PRO 307 16.823 106.361 79.993 1135 CB PRO 307 13.747 105.885 79.833 1136 CG PRO 307 13.190 106.249 81.195 1137 CD PRO 307 13.998 105.463 82.210 1138 N GLN 308 16.572 104.856 78.320 1139 CA GLN 308 17.761 105.207 77.530 1140 C GLN 308 17.871 104.225 76.365 1141 O GLN 308 16.876 103.595 75.993 1142 CB GLN 308 19.024 105.119 78.381 1143 CG GLN 308 19.136 103.785 79.110 1144 CD GLN 308 20.565 103.601 79.596 1145 OE1 GLN 308 21.467 103.362 78.787 1146 NE2 GLN 308 20.746 103.666 80.903 1147 N GLU 309 19.016 104.194 75.712 1148 CA GLU 309 19.273 103.106 74.766 1149 C GLU 309 20.543 102.378 75.191 1150 O GLU 309 21.655 102.857 74.942 1151 CB GLU 309 19.381 103.637 73.339 1152 CG GLU 309 18.059 104.202 72.809 1153 CD GLU 309 16.956 103.142 72.696 1154 OE1 GLU 309 15.833 103.539 72.399 1155 OE2 GLU 309 17.277 101.962 72.718 1156 N PRO 310 20.350 101.179 75.720 1157 CA PRO 310 21.363 100.506 76.543 1158 C PRO 310 22.671 100.273 75.800 1159 O PRO 310 22.695 100.250 74.565 1160 CB PRO 310 20.741 99.219 76.975 1161 CG PRO 310 19.298 99.186 76.508 1162 CD PRO 310 19.066 100.485 75.759 1163 N ALA 311 23.750 100.323 76.567 1164 CA ALA 311 25.135 100.213 76.067 1165 C ALA 311 25.592 101.445 75.272 1166 O ALA 311 26.625 101.415 74.593 1167 CB ALA 311 25.325 98.931 75.261 1168 N PHE 312 24.796 102.497 75.349 1169 CA PHE 312 25.184 103.841 74.943 1170 C PHE 312 24.699 104.711 76.093 1171 O PHE 312 23.573 104.531 76.564 1172 CB PHE 312 24.555 104.208 73.605 1173 CG PHE 312 25.595 104.690 72.597 1174 CD1 PHE 312 25.203 105.425 71.484 1175 CD2 PHE 312 26.934 104.383 72.789 1176 CE1 PHE 312 26.163 105.859 70.570 1177 CE2 PHE 312 27.887 104.815 71.883 1178 CZ PHE 312 27.506 105.553 70.775 1179 N GLU 313 25.547 105.639 76.513 1180 CA GLU 313 25.549 106.156 77.899 1181 C GLU 313 26.098 105.007 78.740 1182 O GLU 313 25.571 104.655 79.801 1183 CB GLU 313 24.167 106.566 78.432 1184 CG GLU 313 23.489 107.664 77.619 1185 CD GLU 313 24.297 108.952 77.696 1186 OE1 GLU 313 25.129 109.150 76.819 1187 OE2 GLU 313 24.112 109.685 78.657 1188 N LEU 314 27.162 104.420 78.211 1189 CA LEU 314 27.739 103.197 78.765 1190 C LEU 314 28.565 103.541 79.996 1191 O LEU 314 29.507 104.342 79.945 1192 CB LEU 314 28.579 102.557 77.656 1193 CG LEU 314 28.842 101.060 77.840 1194 CD1 LEU 314 29.199 100.397 76.513 1195 CD2 LEU 314 29.916 100.767 78.879 1196 N ASP 315 28.108 103.019 81.117 1197 CA ASP 315 28.766 103.246 82.399 1198 C ASP 315 30.034 102.400 82.530 1199 O ASP 315 30.097 101.255 82.056 1200 CB ASP 315 27.734 102.892 83.457 1201 CG ASP 315 28.193 103.229 84.867 1202 OD1 ASP 315 28.799 102.357 85.486 1203 OD2 ASP 315 27.920 104.329 85.320 1204 N TRP 316 30.985 102.924 83.289 1205 CA TRP 316 32.304 102.300 83.440 1206 C TRP 316 32.307 100.924 84.116 1207 O TRP 316 33.140 100.100 83.725 1208 CB TRP 316 33.211 103.250 84.226 1209 CG TRP 316 33.036 103.336 85.732 1210 CD1 TRP 316 31.885 103.570 86.452 1211 CD2 TRP 316 34.101 103.202 86.696 1212 NE1 TRP 316 32.193 103.556 87.773 1213 CE2 TRP 316 33.510 103.342 87.961 1214 CE3 TRP 316 35.464 102.974 86.579 1215 CZ2 TRP 316 34.295 103.250 89.100 1216 CZ3 TRP 316 36.245 102.887 87.724 1217 CH2 TRP 316 35.662 103.023 88.979 1218 N SER 317 31.253 100.567 84.836 1219 CA SER 317 31.191 99.238 85.451 1220 C SER 317 30.895 98.126 84.441 1221 O SER 317 31.387 97.005 84.618 1222 CB SER 317 30.098 99.263 86.509 1223 OG SER 317 29.968 97.951 87.040 1224 N ASP 318 30.314 98.475 83.300 1225 CA ASP 318 30.083 97.469 82.259 1226 C ASP 318 31.386 97.189 81.548 1227 O ASP 318 31.882 96.053 81.523 1228 CB ASP 318 29.158 98.013 81.175 1229 CG ASP 318 27.791 98.391 81.720 1230 OD1 ASP 318 27.014 97.486 81.966 1231 OD2 ASP 318 27.573 99.561 81.995 1232 N ARG 319 32.054 98.280 81.219 1233 CA ARG 319 33.183 98.181 80.315 1234 C ARG 319 34.448 97.720 81.015 1235 O ARG 319 35.096 96.812 80.488 1236 CB ARG 319 33.401 99.530 79.668 1237 CG ARG 319 34.282 99.338 78.453 1238 CD ARG 319 34.531 100.655 77.752 1239 NE ARG 319 33.261 101.278 77.373 1240 CZ ARG 319 33.204 102.587 77.166 1241 NH1 ARG 319 34.298 103.315 77.361 1242 NH2 ARG 319 32.060 103.171 76.805 1243 N LYS 320 34.571 98.031 82.293 1244 CA LYS 320 35.735 97.571 83.052 1245 C LYS 320 35.662 96.071 83.335 1246 O LYS 320 36.681 95.383 83.199 1247 CB LYS 320 35.773 98.348 84.361 1248 CG LYS 320 36.916 97.905 85.263 1249 CD LYS 320 36.917 98.718 86.550 1250 CE LYS 320 35.568 98.622 87.251 1251 NZ LYS 320 35.557 99.430 88.479 1252 N ALA 321 34.455 95.536 83.411 1253 CA ALA 321 34.318 94.101 83.642 1254 C ALA 321 34.474 93.290 82.352 1255 O ALA 321 35.174 92.270 82.367 1256 CB ALA 321 32.959 93.845 84.282 1257 N ILE 322 34.073 93.848 81.217 1258 CA ILE 322 34.234 93.099 79.959 1259 C ILE 322 35.652 93.245 79.387 1260 O ILE 322 36.194 92.270 78.842 1261 CB ILE 322 33.150 93.515 78.958 1262 CG1 ILE 322 33.235 92.702 77.673 1263 CG2 ILE 322 33.192 95.002 78.630 1264 CD1 ILE 322 33.049 91.214 77.944 1265 N GLN 323 36.343 94.297 79.800 1266 CA GLN 323 37.751 94.468 79.443 1267 C GLN 323 38.669 93.742 80.421 1268 O GLN 323 39.845 93.515 80.115 1269 CB GLN 323 38.074 95.954 79.429 1270 CG GLN 323 37.274 96.658 78.342 1271 CD GLN 323 37.541 98.157 78.377 1272 OE1 GLN 323 37.702 98.755 79.449 1273 NE2 GLN 323 37.488 98.762 77.204 1274 N LYS 324 38.098 93.251 81.509 1275 CA LYS 324 38.838 92.394 82.432 1276 C LYS 324 38.782 90.941 81.962 1277 O LYS 324 39.670 90.141 82.282 1278 CB LYS 324 38.186 92.543 83.802 1279 CG LYS 324 38.970 91.858 84.911 1280 CD LYS 324 38.332 92.156 86.260 1281 CE LYS 324 38.226 93.661 86.491 1282 NZ LYS 324 37.604 93.961 87.792 1283 N LEU 325 37.838 90.657 81.076 1284 CA LEU 325 37.749 89.330 80.467 1285 C LEU 325 38.660 89.197 79.254 1286 O LEU 325 39.629 88.431 79.286 1287 CB LEU 325 36.315 89.089 80.027 1288 CG LEU 325 35.390 88.884 81.216 1289 CD1 LEU 325 33.943 88.992 80.773 1290 CD2 LEU 325 35.658 87.541 81.886 1291 N TYR 326 38.353 89.935 78.199 1292 CA TYR 326 39.109 89.764 76.950 1293 C TYR 326 40.023 90.938 76.619 1294 O TYR 326 40.552 91.002 75.504 1295 CB TYR 326 38.148 89.568 75.786 1296 CG TYR 326 37.137 88.442 75.962 1297 CD1 TYR 326 37.558 87.122 76.072 1298 CD2 TYR 326 35.785 88.749 76.012 1299 CE1 TYR 326 36.623 86.107 76.228 1300 CE2 TYR 326 34.852 87.737 76.167 1301 CZ TYR 326 35.272 86.419 76.273 1302 OH TYR 326 34.336 85.421 76.426 1303 N GLY 327 40.157 91.883 77.532 1304 CA GLY 327 41.002 93.048 77.261 1305 C GLY 327 40.217 94.229 76.697 1306 O GLY 327 39.082 94.100 76.218 1307 N SER 328 40.821 95.394 76.844 1308 CA SER 328 40.293 96.634 76.268 1309 C SER 328 40.692 96.743 74.804 1310 O SER 328 41.735 96.184 74.449 1311 CB SER 328 40.919 97.797 77.038 1312 OG SER 328 40.478 99.022 76.462 1313 N CYS 329 39.770 97.242 73.982 1314 CA CYS 329 40.009 97.698 72.584 1315 C CYS 329 38.942 97.288 71.581 1316 O CYS 329 37.904 96.696 71.898 1317 CB CYS 329 41.360 97.333 71.967 1318 SG CYS 329 42.744 98.423 72.379 1319 N GLU 330 39.368 97.517 70.348 1320 CA GLU 330 38.622 97.415 69.080 1321 C GLU 330 38.276 98.844 68.701 1322 O GLU 330 37.113 99.259 68.650 1323 CB GLU 330 37.376 96.547 69.121 1324 CG GLU 330 36.937 96.178 67.708 1325 CD GLU 330 37.957 95.234 67.092 1326 OE1 GLU 330 38.045 94.110 67.569 1327 OE2 GLU 330 38.788 95.727 66.340

TABLE VI Atom Atom Residue No Name Residue No X coord Y coord Z coord 1 N GLN 346 31.981 116.323 91.895 2 CA GLN 346 31.747 117.708 91.457 3 C GLN 346 30.383 117.854 90.782 4 O GLN 346 30.162 117.329 89.685 5 CB GLN 346 32.844 118.087 90.473 6 CG GLN 346 34.223 117.902 91.094 7 CD GLN 346 35.302 118.254 90.079 8 OE1 GLN 346 36.464 118.484 90.439 9 NE2 GLN 346 34.896 118.314 88.822 10 N TYR 347 29.502 118.606 91.419 11 CA TYR 347 28.129 118.765 90.913 12 C TYR 347 27.909 120.137 90.283 13 O TYR 347 28.820 120.975 90.248 14 CB TYR 347 27.144 118.558 92.061 15 CG TYR 347 27.180 117.164 92.686 16 CD1 TYR 347 28.046 116.889 93.737 17 CD2 TYR 347 26.340 116.169 92.208 18 CE1 TYR 347 28.081 115.619 94.290 19 CE2 TYR 347 26.372 114.897 92.764 20 CZ TYR 347 27.244 114.625 93.809 21 OH TYR 347 27.246 113.388 94.414 22 N GLY 348 26.711 120.337 89.759 23 CA GLY 348 26.358 121.630 89.158 24 C GLY 348 25.055 122.192 89.721 25 O GLY 348 24.944 123.398 89.984 26 N GLU 349 24.133 121.300 90.029 27 CA GLU 349 22.818 121.717 90.533 28 C GLU 349 22.418 121.009 91.834 29 O GLU 349 22.411 119.774 91.881 30 CB GLU 349 21.833 121.413 89.414 31 CG GLU 349 22.258 120.174 88.636 32 CD GLU 349 21.193 119.714 87.646 33 OE1 GLU 349 20.114 119.403 88.141 34 OE2 GLU 349 21.606 119.251 86.591 35 N VAL 350 22.152 121.784 92.884 36 CA VAL 350 21.776 121.222 94.204 37 C VAL 350 20.769 122.099 94.969 38 O VAL 350 21.187 122.972 95.741 39 CB VAL 350 23.014 121.072 95.100 40 CG1 VAL 350 22.648 120.486 96.462 41 CG2 VAL 350 24.104 120.218 94.468 42 N MET 351 19.479 121.840 94.779 43 CA MET 351 18.390 122.507 95.543 44 C MET 351 17.046 121.789 95.389 45 O MET 351 16.668 121.470 94.258 46 CB MET 351 18.151 123.941 95.056 47 CG MET 351 19.049 124.989 95.703 48 SD MET 351 18.619 126.705 95.346 49 CE MET 351 17.090 126.824 96.301 50 N VAL 352 16.344 121.556 96.496 51 CA VAL 352 14.927 121.106 96.462 52 C VAL 352 14.159 121.521 97.722 53 O VAL 352 14.591 122.390 98.484 54 CB VAL 352 14.730 119.603 96.237 55 CG1 VAL 352 14.806 119.177 94.777 56 CG2 VAL 352 15.609 118.740 97.115 57 N ARG 353 12.987 120.920 97.887 58 CA ARG 353 12.070 121.237 98.996 59 C ARG 353 12.492 120.590 100.314 60 O ARG 353 12.086 119.452 100.585 61 CB ARG 353 10.705 120.674 98.618 62 CG ARG 353 10.351 121.062 97.190 63 CD ARG 353 9.004 120.515 96.737 64 NE ARG 353 8.733 120.973 95.366 65 CZ ARG 353 7.782 120.460 94.582 66 NH1 ARG 353 7.036 119.439 95.010 67 NH2 ARG 353 7.603 120.950 93.355 68 N PHE 354 13.301 121.307 101.094 69 CA PHE 354 13.872 120.866 102.401 70 C PHE 354 14.986 119.816 102.274 71 O PHE 354 16.010 119.885 102.965 72 CB PHE 354 12.786 120.299 103.316 73 CG PHE 354 11.785 121.300 103.884 74 CD1 PHE 354 12.223 122.333 104.702 75 CD2 PHE 354 10.431 121.161 103.605 76 CE1 PHE 354 11.309 123.234 105.233 77 CE2 PHE 354 9.517 122.063 104.134 78 CZ PHE 354 9.956 123.099 104.948 79 N SER 355 14.742 118.824 101.439 80 CA SER 355 15.708 117.796 101.077 81 C SER 355 16.856 118.415 100.281 82 O SER 355 16.733 119.515 99.730 83 CB SER 355 14.930 116.790 100.229 84 OG SER 355 15.774 115.717 99.857 85 N THR 356 17.988 117.739 100.275 86 CA THR 356 19.141 118.217 99.516 87 C THR 356 19.408 117.298 98.327 88 O THR 356 19.884 116.173 98.499 89 CB THR 356 20.339 118.214 100.452 90 OG1 THR 356 19.912 118.729 101.706 91 CG2 THR 356 21.476 119.073 99.917 92 N TYR 357 19.064 117.763 97.138 93 CA TYR 357 19.280 116.949 95.928 94 C TYR 357 20.508 117.387 95.146 95 O TYR 357 20.530 118.480 94.567 96 CB TYR 357 18.085 117.034 94.987 97 CG TYR 357 16.895 116.124 95.283 98 CD1 TYR 357 16.818 115.405 96.469 99 CD2 TYR 357 15.888 116.002 94.333 100 CE1 TYR 357 15.714 114.602 96.721 101 CE2 TYR 357 14.786 115.201 94.583 102 CZ TYR 357 14.697 114.509 95.783 103 OH TYR 357 13.575 113.763 96.062 104 N PHE 358 21.469 116.486 95.058 105 CA PHE 358 22.681 116.717 94.268 106 C PHE 358 22.531 116.094 92.877 107 O PHE 358 22.048 114.963 92.733 108 CB PHE 358 23.872 116.093 94.994 109 CG PHE 358 24.160 116.632 96.396 110 CD1 PHE 358 23.619 116.012 97.514 111 CD2 PHE 358 24.999 117.728 96.552 112 CE1 PHE 358 23.879 116.510 98.782 113 CE2 PHE 358 25.259 118.229 97.822 114 CZ PHE 358 24.694 117.622 98.935 115 N PHE 359 22.808 116.894 91.859 116 CA PHE 359 22.753 116.402 90.472 117 C PHE 359 24.025 116.701 89.670 118 O PHE 359 24.591 117.808 89.720 119 CB PHE 359 21.605 117.064 89.723 120 CG PHE 359 20.155 116.709 90.043 121 CD1 PHE 359 19.373 116.136 89.050 122 CD2 PHE 359 19.595 117.010 91.277 123 CE1 PHE 359 18.045 115.829 89.301 124 CE2 PHE 359 18.267 116.696 91.532 125 CZ PHE 359 17.494 116.103 90.543 126 N ARG 360 24.388 115.720 88.856 127 CA ARG 360 25.512 115.840 87.909 128 C ARG 360 25.422 114.720 86.875 129 O ARG 360 24.911 113.644 87.197 130 CB ARG 360 26.854 115.825 88.650 131 CG ARG 360 27.214 114.502 89.325 132 CD ARG 360 28.130 113.651 88.454 133 NE ARG 360 29.245 114.472 87.961 134 CZ ARG 360 30.506 114.043 87.885 135 NH1 ARG 360 30.812 112.800 88.262 136 NH2 ARG 360 31.458 114.856 87.424 137 N ASN 361 26.068 114.903 85.732 138 CA ASN 361 25.926 113.979 84.585 139 C ASN 361 24.483 113.522 84.416 140 O ASN 361 23.569 114.347 84.296 141 CB ASN 361 26.798 112.738 84.776 142 CG ASN 361 28.287 113.047 84.656 143 OD1 ASN 361 28.688 114.180 84.364 144 ND2 ASN 361 29.088 112.013 84.838 145 N SER 362 24.301 112.211 84.421 146 CA SER 362 22.971 111.595 84.302 147 C SER 362 22.400 111.132 85.650 148 O SER 362 21.483 110.302 85.662 149 CB SER 362 23.068 110.393 83.367 150 OG SER 362 23.913 109.423 83.976 151 N TRP 363 22.957 111.625 86.746 152 CA TRP 363 22.572 111.208 88.104 153 C TRP 363 21.635 112.190 88.812 154 O TRP 363 21.883 113.404 88.830 155 CB TRP 363 23.858 111.118 88.913 156 CG TRP 363 24.836 110.088 88.397 157 CD1 TRP 363 26.080 110.300 87.844 158 CD2 TRP 363 24.624 108.665 88.404 159 NE1 TRP 363 26.624 109.094 87.538 160 CE2 TRP 363 25.789 108.090 87.870 161 CE3 TRP 363 23.578 107.865 88.839 162 CZ2 TRP 363 25.897 106.712 87.797 163 CZ3 TRP 363 23.694 106.486 88.758 164 CH2 TRP 363 24.851 105.914 88.243 165 N TYR 364 20.603 111.646 89.445 166 CA TYR 364 19.666 112.472 90.223 167 C TYR 364 19.674 112.126 91.705 168 O TYR 364 20.129 111.042 92.108 169 CB TYR 364 18.233 112.388 89.675 170 CG TYR 364 17.304 111.181 89.913 171 CD1 TYR 364 17.662 110.072 90.670 172 CD2 TYR 364 16.033 111.236 89.353 173 CE1 TYR 364 16.763 109.029 90.849 174 CE2 TYR 364 15.136 110.192 89.523 175 CZ TYR 364 15.505 109.087 90.270 176 OH TYR 364 14.646 108.017 90.380 177 N TRP 365 19.185 113.087 92.483 178 CA TRP 365 18.942 112.983 93.942 179 C TRP 365 20.200 113.021 94.806 180 O TRP 365 20.331 113.906 95.661 181 CB TRP 365 18.202 111.692 94.290 182 CG TRP 365 16.703 111.633 94.048 183 CD1 TRP 365 15.980 112.090 92.972 184 CD2 TRP 365 15.749 111.046 94.958 185 NE1 TRP 365 14.674 111.798 93.172 186 CE2 TRP 365 14.492 111.173 94.349 187 CE3 TRP 365 15.869 110.429 96.196 188 CZ2 TRP 365 13.360 110.683 94.991 189 CZ3 TRP 365 14.732 109.947 96.831 190 CH2 TRP 365 13.485 110.071 96.232 191 N LEU 366 21.099 112.085 94.543 192 CA LEU 366 22.322 111.842 95.310 193 C LEU 366 22.131 112.083 96.789 194 O LEU 366 22.487 113.153 97.296 195 CB LEU 366 23.492 112.668 94.789 196 CG LEU 366 24.229 111.949 93.666 197 CD1 LEU 366 24.259 110.459 93.969 198 CD2 LEU 366 23.610 112.188 92.293 199 N TYR 367 21.674 111.033 97.456 200 CA TYR 367 21.398 111.023 98.904 201 C TYR 367 20.799 112.335 99.402 202 O TYR 367 21.518 113.211 99.894 203 CB TYR 367 22.653 110.623 99.688 204 CG TYR 367 24.022 111.048 99.141 205 CD1 TYR 367 24.772 110.152 98.386 206 CD2 TYR 367 24.532 112.312 99.417 207 CE1 TYR 367 26.016 110.524 97.892 208 CE2 TYR 367 25.775 112.684 98.924 209 CZ TYR 367 26.515 111.790 98.163 210 OH TYR 367 27.760 112.158 97.696 211 N GLU 368 19.484 112.283 99.564 212 CA GLU 368 18.651 113.475 99.787 213 C GLU 368 18.725 114.134 101.178 214 O GLU 368 17.993 115.104 101.408 215 CB GLU 368 17.213 113.085 99.416 216 CG GLU 368 16.695 111.779 100.025 217 CD GLU 368 16.389 111.958 101.514 218 OE1 GLU 368 17.251 111.487 102.241 219 OE2 GLU 368 15.741 112.953 101.768 220 N ASN 369 19.531 113.594 102.086 221 CA ASN 369 19.811 114.158 103.429 222 C ASN 369 18.678 114.063 104.480 223 O ASN 369 18.963 113.757 105.647 224 CB ASN 369 20.271 115.600 103.205 225 CG ASN 369 20.455 116.364 104.504 226 OD1 ASN 369 19.694 117.294 104.793 227 ND2 ASN 369 21.490 116.009 105.245 228 N ARG 370 17.428 114.255 104.089 229 CA ARG 370 16.291 114.087 105.004 230 C ARG 370 16.038 112.626 105.370 231 O ARG 370 15.539 111.845 104.551 232 CB ARG 370 15.037 114.636 104.331 233 CG ARG 370 14.908 116.143 104.485 234 CD ARG 370 14.743 116.486 105.960 235 NE ARG 370 14.373 117.893 106.163 236 CZ ARG 370 13.209 118.250 106.711 237 NH1 ARG 370 12.288 117.323 106.982 238 NH2 ARG 370 12.936 119.541 106.918 239 N ASN 371 16.355 112.309 106.617 240 CA ASN 371 16.098 110.992 107.236 241 C ASN 371 16.408 109.818 106.314 242 O ASN 371 15.495 109.166 105.790 243 CB ASN 371 14.632 110.938 107.656 244 CG ASN 371 14.356 111.970 108.747 245 OD1 ASN 371 14.061 113.138 108.469 246 ND2 ASN 371 14.443 111.513 109.984 247 N ASN 372 17.686 109.541 106.135 248 CA ASN 372 18.067 108.499 105.184 249 C ASN 372 18.982 107.444 105.807 250 O ASN 372 19.951 107.763 106.505 251 CB ASN 372 18.758 109.188 104.017 252 CG ASN 372 18.877 108.224 102.848 253 OD1 ASN 372 18.180 107.203 102.795 254 ND2 ASN 372 19.821 108.506 101.972 255 N ARG 373 18.655 106.190 105.534 256 CA ARG 373 19.463 105.056 105.995 257 C ARG 373 20.370 104.514 104.888 258 O ARG 373 21.339 103.803 105.176 259 CB ARG 373 18.533 103.931 106.446 260 CG ARG 373 17.696 104.328 107.653 261 CD ARG 373 18.582 104.684 108.843 262 NE ARG 373 17.764 105.092 109.994 263 CZ ARG 373 17.530 106.369 110.303 264 NH1 ARG 373 16.777 106.671 111.362 265 NH2 ARG 373 18.056 107.344 109.558 266 N THR 374 20.063 104.838 103.640 267 CA THR 374 20.840 104.292 102.514 268 C THR 374 20.925 105.301 101.373 269 O THR 374 19.892 105.737 100.857 270 CB THR 374 20.134 103.031 102.023 271 OG1 THR 374 20.004 102.138 103.122 272 CG2 THR 374 20.904 102.317 100.916 273 N ARG 375 22.131 105.684 100.989 274 CA ARC 375 22.278 106.695 99.931 275 C ARG 375 21.678 106.251 98.595 276 O ARG 375 21.873 105.127 98.118 277 CB ARG 375 23.745 107.069 99.766 278 CG ARG 375 24.646 105.861 99.561 279 CD ARC 375 26.075 106.300 99.259 280 NE ARG 375 26.582 107.215 100.296 281 CZ ARG 375 27.797 107.107 100.839 282 NH1 ARG 375 28.205 108.002 101.741 283 NH2 ARG 375 28.614 106.123 100.458 284 N TYR 376 20.873 107.148 98.052 285 CA TYR 376 20.185 106.904 96.777 286 C TYR 376 20.949 107.581 95.641 287 O TYR 376 21.250 108.776 95.730 288 CB TYR 376 18.776 107.492 96.869 289 CG TYR 376 17.966 107.036 98.086 290 CD1 TYR 376 17.832 105.684 98.373 291 CD2 TYR 376 17.346 107.980 98.896 292 CE1 TYR 376 17.109 105.276 99.486 293 CE2 TYR 376 16.620 107.574 100.008 294 CZ TYR 376 16.513 106.222 100.306 295 OH TYR 376 15.925 105.825 101.487 296 N GLY 377 21.194 106.855 94.562 297 CA GLY 377 21.969 107.417 93.443 298 C GLY 377 21.582 106.774 92.115 299 O GLY 377 22.265 105.871 91.616 300 N ASP 378 20.504 107.275 91.534 301 CA ASP 378 19.942 106.655 90.327 302 C ASP 378 19.990 107.645 89.158 303 O ASP 378 20.162 108.845 89.389 304 CB ASP 378 18.481 106.275 90.595 305 CG ASP 378 18.203 105.814 92.032 306 OD1 ASP 378 18.975 105.029 92.570 307 OD2 ASP 378 17.233 106.310 92.592 308 N PRO 379 19.973 107.151 87.931 309 CA PRO 379 19.783 108.031 86.765 310 C PRO 379 18.486 108.851 86.842 311 O PRO 379 17.487 108.410 87.426 312 CB PRO 379 19.835 107.126 85.576 313 CG PRO 379 20.042 105.692 86.042 314 CD PRO 379 20.066 105.737 87.560 315 N ILE 380 18.485 109.998 86.174 316 CA ILE 380 17.442 111.026 86.392 317 C ILE 380 16.060 110.799 85.762 318 O ILE 380 15.104 111.465 86.169 319 CB ILE 380 17.937 112.368 85.855 320 CG1 ILE 380 19.414 112.583 86.109 321 CG2 ILE 380 17.179 113.510 86.519 322 CD1 ILE 380 19.878 113.937 85.584 323 N GLN 381 15.895 109.837 84.876 324 CA GLN 381 14.610 109.793 84.166 325 C GLN 381 13.547 108.878 84.788 326 O GLN 381 12.485 108.680 84.187 327 CB GLN 381 14.850 109.524 82.686 328 CG GLN 381 15.641 110.703 82.115 329 CD GLN 381 15.660 110.704 80.590 330 OE1 GLN 381 15.567 109.650 79.951 331 NE2 GLN 381 15.775 111.896 80.026 332 N ILE 382 13.799 108.352 85.977 333 CA ILE 382 12.732 107.644 86.696 334 C ILE 382 11.876 108.673 87.443 335 O ILE 382 12.408 109.496 88.194 336 CB ILE 382 13.357 106.596 87.615 337 CG1 ILE 382 13.990 105.500 86.768 338 CG2 ILE 382 12.340 105.990 88.578 339 CD1 ILE 382 12.960 104.866 85.837 340 N LEU 383 10.574 108.629 87.172 341 CA LEU 383 9.557 109.617 87.616 342 C LEU 383 9.447 109.931 89.115 343 O LEU 383 8.452 109.559 89.745 344 CB LEU 383 8.192 109.061 87.218 345 CG LEU 383 8.021 108.889 85.716 346 CD1 LEU 383 6.834 107.981 85.406 347 CD2 LEU 383 7.872 110.236 85.021 348 N THR 384 10.344 110.756 89.629 349 CA THR 384 10.218 111.231 91.012 350 C THR 384 9.736 112.679 91.090 351 O THR 384 9.710 113.266 92.175 352 CB THR 384 11.545 111.077 91.746 353 OG1 THR 384 12.528 111.918 91.153 354 CG2 THR 384 12.033 109.634 91.700 355 N GLY 385 9.420 113.264 89.948 356 CA GLY 385 8.862 114.618 89.952 357 C GLY 385 8.985 115.332 88.612 358 O GLY 385 8.322 116.355 88.383 359 N TRP 386 9.848 114.810 87.755 360 CA TRP 386 10.132 115.486 86.482 361 C TRP 386 9.988 114.539 85.299 362 O TRP 386 10.983 113.959 84.847 363 CB TRP 386 11.576 115.981 86.508 364 CG TRP 386 12.064 116.393 87.880 365 CD1 TRP 386 11.738 117.536 88.574 366 CD2 TRP 386 12.969 115.648 88.723 367 NE1 TRP 386 12.360 117.498 89.777 368 CE2 TRP 386 13.090 116.379 89.916 369 CE3 TRP 386 13.629 114.439 88.573 370 CZ2 TRP 386 13.848 115.877 90.961 371 CZ3 TRP 386 14.398 113.951 89.620 372 CH2 TRP 386 14.503 114.663 90.810 373 N PRO 387 8.792 114.483 84.728 374 CA PRO 387 8.511 113.566 83.613 375 C PRO 387 9.115 113.989 82.265 376 O PRO 387 9.055 113.219 81.301 377 CB PRO 387 7.016 113.532 83.519 378 CG PRO 387 6.428 114.603 84.424 379 CD PRO 387 7.607 115.240 85.140 380 N GLY 388 9.713 115.167 82.208 381 CA GLY 388 10.375 115.624 80.986 382 C GLY 388 11.661 116.373 81.325 383 O GLY 388 11.967 117.398 80.707 384 N ILE 389 12.393 115.843 82.292 385 CA ILE 389 13.668 116.425 82.736 386 C ILE 389 14.658 116.458 81.560 387 O ILE 389 14.612 115.580 80.689 388 CB ILE 389 14.131 115.537 83.910 389 CG1 ILE 389 15.265 116.108 84.768 390 CG2 ILE 389 14.491 114.136 83.427 391 CD1 ILE 389 16.661 115.865 84.198 392 N PRO 390 15.466 117.510 81.489 393 CA PRO 390 16.485 117.642 80.440 394 C PRO 390 17.381 116.415 80.305 395 O PRO 390 17.445 115.540 81.180 396 CB PRO 390 17.300 118.840 80.822 397 CG PRO 390 16.685 119.498 82.045 398 CD PRO 390 15.476 118.655 82.409 399 N THR 391 18.005 116.318 79.146 400 CA THR 391 18.955 115.237 78.908 401 C THR 391 20.305 115.558 79.547 402 O THR 391 21.166 116.216 78.948 403 CB THR 391 19.068 115.000 77.404 404 OG1 THR 391 20.217 114.196 77.170 405 CG2 THR 391 19.229 116.299 76.618 406 N HIS 392 20.442 115.072 80.776 407 CA HIS 392 21.645 115.183 81.620 408 C HIS 392 22.434 116.491 81.497 409 O HIS 392 21.872 117.546 81.174 410 CB HIS 392 22.547 113.963 81.380 411 CG HIS 392 22.739 113.479 79.951 412 ND1 HIS 392 22.165 112.396 79.391 413 CD2 HIS 392 23.552 114.037 78.994 414 CE1 HIS 392 22.571 112.289 78.110 415 NE2 HIS 392 23.429 113.304 77.865 416 N ASN 393 23.613 116.449 82.097 417 CA ASN 393 24.643 117.500 81.979 418 C ASN 393 24.461 118.687 82.916 419 O ASN 393 23.983 119.742 82.476 420 CB ASN 393 24.742 118.047 80.559 421 CG ASN 393 25.250 117.004 79.577 422 OD1 ASN 393 26.029 116.111 79.931 423 ND2 ASN 393 24.763 117.118 78.354 424 N ILE 394 24.712 118.416 84.192 425 CA ILE 394 25.113 119.335 85.301 426 C ILE 394 24.505 120.744 85.557 427 O ILE 394 24.471 121.131 86.731 428 CB ILE 394 26.622 119.486 85.061 429 CG1 ILE 394 27.283 118.113 85.033 430 CG2 ILE 394 27.315 120.363 86.094 431 CD1 ILE 394 28.780 118.204 84.763 432 N ASP 395 23.927 121.427 84.584 433 CA ASP 395 23.584 122.855 84.710 434 C ASP 395 22.489 123.170 85.720 435 O ASP 395 21.770 122.287 86.212 436 CB ASP 395 23.166 123.409 83.356 437 CG ASP 395 24.286 123.313 82.329 438 OD1 ASP 395 23.953 123.369 81.154 439 OD2 ASP 395 25.448 123.341 82.720 440 N ALA 396 22.377 124.469 85.961 441 CA ALA 396 21.535 125.084 86.999 442 C ALA 396 20.181 124.439 87.277 443 O ALA 396 19.530 123.829 86.418 444 CB ALA 396 21.324 126.546 86.624 445 N PHE 397 19.789 124.583 88.529 446 CA PHE 397 18.529 124.034 89.026 447 C PHE 397 18.071 124.851 90.228 448 O PHE 397 18.307 124.473 91.383 449 CB PHE 397 18.773 122.585 89.421 450 CG PHE 397 17.592 121.786 89.958 451 CD1 PHE 397 17.826 120.727 90.824 452 CD2 PHE 397 16.293 122.101 89.579 453 CE1 PHE 397 16.764 119.983 91.315 454 CE2 PHE 397 15.230 121.358 90.073 455 CZ PHE 397 15.466 120.298 90.940 456 N VAL 398 17.440 125.972 89.934 457 CA VAL 398 16.972 126.874 90.986 458 C VAL 398 15.641 126.388 91.542 459 O VAL 398 14.813 125.813 90.825 460 CB VAL 398 16.879 128.296 90.423 461 CG1 VAL 398 16.171 128.355 89.082 462 CG2 VAL 398 16.270 129.292 91.401 463 N HIS 399 15.520 126.455 92.854 464 CA HIS 399 14.263 126.075 93.474 465 C HIS 399 13.551 127.237 94.145 466 O HIS 399 14.066 127.860 95.080 467 CB HIS 399 14.540 125.001 94.500 468 CG HIS 399 13.820 123.724 94.169 469 ND1 HIS 399 14.161 122.848 93.213 470 CD2 HIS 399 12.686 123.248 94.771 471 CE1 HIS 399 13.282 121.827 93.221 472 NE2 HIS 399 12.370 122.075 94.188 473 N ILE 400 12.323 127.448 93.711 474 CA ILE 400 11.423 128.387 94.380 475 C ILE 400 10.290 127.552 94.971 476 O ILE 400 9.094 127.767 94.732 477 CB ILE 400 10.902 129.424 93.389 478 CG1 ILE 400 12.048 129.995 92.560 479 CG2 ILE 400 10.212 130.561 94.131 480 CD1 ILE 400 11.559 131.091 91.622 481 N TRP 401 10.707 126.625 95.818 482 CA TRP 401 9.825 125.602 96.387 483 C TRP 401 8.982 126.075 97.552 484 O TRP 401 8.079 125.341 97.965 485 CB TRP 401 10.683 124.455 96.899 486 CG TRP 401 11.727 124.819 97.943 487 CD1 TRP 401 13.031 125.202 97.708 488 CD2 TRP 401 11.559 124.811 99.380 489 NE1 TRP 401 13.635 125.439 98.899 490 CE2 TRP 401 12.788 125.223 99.923 491 CE3 TRP 401 10.493 124.508 100.210 492 CZ2 TRP 401 12.928 125.341 101.298 493 CZ3 TRP 401 10.642 124.624 101.587 494 CH2 TRP 401 11.855 125.041 102.127 495 N THR 402 9.208 127.305 97.993 496 CA THR 402 8.504 127.887 99.140 497 C THR 402 7.013 127.618 99.087 498 O THR 402 6.426 127.540 98.001 499 CB THR 402 8.718 129.392 99.111 500 OG1 THR 402 8.113 129.890 97.922 501 CG2 THR 402 10.200 129.745 99.088 502 N TRP 403 6.397 127.725 100.250 503 CA TRP 403 4.970 127.432 100.474 504 C TRP 403 3.973 128.419 99.844 505 O TRP 403 2.765 128.315 100.079 506 CB TRP 403 4.763 127.374 101.991 507 CG TRP 403 5.789 128.158 102.798 508 CD1 TRP 403 6.817 127.625 103.547 509 CD2 TRP 403 5.877 129.594 102.951 510 NE1 TRP 403 7.529 128.643 104.093 511 CE2 TRP 403 7.009 129.838 103.750 512 CE3 TRP 403 5.131 130.653 102.456 513 CZ2 TRP 403 7.387 131.142 104.027 514 CZ3 TRP 403 5.514 131.957 102.744 515 CH2 TRP 403 6.640 132.201 103.526 516 N LYS 404 4.475 129.367 99.069 517 CA LYS 404 3.638 130.295 98.325 518 C LYS 404 3.684 129.983 96.823 519 O LYS 404 2.759 130.347 96.088 520 CB LYS 404 4.214 131.685 98.584 521 CG LYE 404 3.481 132.789 97.832 522 CD LYS 404 4.189 134.126 98.013 523 CE LYS 404 3.516 135.229 97.206 524 NZ LYS 404 4.238 136.503 97.355 525 N ARG 405 4.710 129.262 96.381 526 CA ARG 405 4.925 129.098 94.933 527 C ARG 405 5.127 127.645 94.476 528 O ARG 405 4.572 127.260 93.439 529 CB ARG 405 6.132 129.946 94.530 530 CG ARG 405 5.903 131.421 94.860 531 CD ARG 405 7.038 132.324 94.395 532 NE ARG 405 7.125 132.342 92.927 533 CZ ARG 405 7.141 133.461 92.201 534 NH1 ARG 405 7.126 134.651 92.805 535 NH2 ARG 405 7.209 133.389 90.869 536 N ASP 406 5.875 126.862 95.244 537 CA ASP 406 6.178 125.446 94.931 538 C ASP 406 6.615 125.225 93.475 539 O ASP 406 5.867 124.674 92.658 540 CB ASP 406 4.928 124.625 95.242 541 CG ASP 406 5.218 123.127 95.180 542 OD1 ASP 406 5.840 122.633 96.108 543 OD2 ASP 406 4.820 122.500 94.204 544 N GLU 407 7.801 125.708 93.140 545 CA GLU 407 8.284 125.636 91.749 546 C GLU 407 9.713 125.105 91.604 547 O GLU 407 10.666 125.704 92.124 548 CB GLU 407 8.269 127.057 91.195 549 CG GLU 407 6.885 127.691 91.236 550 CD GLU 407 7.002 129.192 91.001 551 OE1 GLU 407 5.973 129.854 91.016 552 OE2 GLU 407 8.131 129.658 90.926 553 N ARG 408 9.859 124.022 90.858 554 CA ARG 408 11.195 123.555 90.438 555 C ARG 408 11.643 124.258 89.156 556 O ARG 408 10.784 124.720 88.400 557 CB ARG 408 11.110 122.059 90.185 558 CG ARG 408 10.615 121.354 91.438 559 CD ARG 408 10.570 119.847 91.276 560 NE ARG 408 10.678 119.181 92.583 561 CZ ARG 408 10.000 118.079 92.909 562 NH1 ARG 408 9.081 117.591 92.074 563 NH2 ARG 408 10.173 117.526 94.111 564 N TYR 409 12.941 124.412 88.943 565 CA TYR 409 13.430 125.008 87.676 566 C TYR 409 14.760 124.439 87.173 567 O TYR 409 15.832 124.848 87.636 568 CB TYR 409 13.649 126.504 87.853 569 CG TYR 409 12.421 127.399 87.935 570 CD1 TYR 409 11.880 127.737 89.169 571 CD2 TYR 409 11.867 127.908 86.768 572 CE1 TYR 409 10.766 128.562 89.234 573 CE2 TYR 409 10.756 128.737 86.833 574 CZ TYR 409 10.206 129.057 88.066 575 OH TYR 409 9.070 129.832 88.131 576 N PHE 410 14.689 123.654 86.108 577 CA PHE 410 15.903 123.109 85.472 578 C PHE 410 16.436 124.009 84.357 579 O PHE 410 15.687 124.793 83.760 580 CB PHE 410 15.612 121.738 84.880 581 CG PHE 410 15.580 120.600 85.891 582 CD1 PHE 410 16.776 120.050 86.337 583 CD2 PHE 410 14.371 120.110 86.359 584 CE1 PHE 410 16.761 119.009 87.256 585 CE2 PHE 410 14.358 119.070 87.276 586 CZ PHE 410 15.550 118.519 87.726 587 N PHE 411 17.739 123.932 84.137 588 CA PHE 411 18.390 124.670 83.042 589 C PHE 411 19.327 123.781 82.226 590 O PHE 411 20.130 123.018 82.778 591 CB PHE 411 19.215 125.818 83.619 592 CG PHE 411 18.440 127.063 84.043 593 CD1 PHE 411 17.787 127.112 85.270 594 CD2 PHE 411 18.409 128.163 83.197 595 CE1 PHE 411 17.084 128.252 85.636 596 CE2 PHE 411 17.712 129.305 83.567 597 CZ PHE 411 17.045 129.347 84.784 598 N GLN 412 19.268 123.930 80.915 599 CA GLN 412 20.226 123.224 80.055 600 C GLN 412 20.809 124.169 79.009 601 O GLN 412 20.196 124.447 77.971 602 CB GLN 412 19.566 121.995 79.424 603 CG GLN 412 20.474 121.259 78.434 604 CD GLN 412 21.888 121.068 78.987 605 OE1 GLN 412 22.839 121.656 78.460 606 NE2 GLN 412 22.022 120.235 79.999 607 N GLY 413 22.050 124.560 79.259 608 CA GLY 413 22.726 125.561 78.439 609 C GLY 413 21.925 126.850 78.424 610 O GLY 413 21.537 127.385 79.473 611 N ASN 414 21.529 127.212 77.221 612 CA ASN 414 20.747 128.422 76.981 613 C ASN 414 19.244 128.149 76.868 614 O ASN 414 18.646 128.411 75.818 615 CB ASN 414 21.294 129.080 75.713 616 CG ASN 414 21.630 128.073 74.601 617 OD1 ASN 414 22.798 127.725 74.394 618 ND2 ASN 414 20.605 127.592 73.920 619 N GLN 415 18.653 127.599 77.916 620 CA GLN 415 17.192 127.386 77.948 621 C GLN 415 16.723 126.926 79.329 622 O GLN 415 17.298 126.011 79.939 623 CB GLN 415 16.765 126.381 76.873 624 CG GLN 415 17.296 124.980 77.134 625 CD GLN 415 17.374 124.176 75.844 626 OE1 GLN 415 16.374 123.963 75.151 627 NE2 GLN 415 18.579 123.704 75.574 628 N TYR 416 15.740 127.640 79.850 629 CA TYR 416 15.139 127.258 81.132 630 C TYR 416 13.900 126.407 80.898 631 O TYR 416 13.201 126.547 79.886 632 CB TYR 416 14.797 128.486 81.978 633 CG TYR 416 13.679 129.405 81.491 634 CD1 TYR 416 13.918 130.317 80.473 635 CD2 TYR 416 12.429 129.357 82.099 636 CE1 TYR 416 12.903 131.155 80.038 637 CE2 TYR 416 11.411 130.198 81.666 638 CZ TYR 416 11.653 131.091 80.631 639 OH TYR 416 10.635 131.872 80.136 640 N TRP 417 13.684 125.482 81.814 641 CA TRP 417 12.546 124.576 81.708 642 C TRP 417 11.231 125.271 82.017 643 O TRP 417 11.187 126.276 82.739 644 CB TRP 417 12.779 123.339 82.558 645 CG TRP 417 13.004 122.135 81.670 646 CD1 TRP 417 12.237 120.994 81.636 647 CD2 TRP 417 14.048 121.958 80.683 648 NE1 TRP 417 12.718 120.183 80.661 649 CE2 TRP 417 13.777 120.737 80.043 650 CE3 TRP 417 15.108 122.747 80.260 651 CZ2 TRP 417 14.539 120.347 78.950 652 CZ3 TRP 417 15.882 122.336 79.182 653 CH2 TRP 417 15.595 121.144 78.526 654 N ARG 418 10.177 124.647 81.511 655 CA ARG 418 8.844 125.247 81.335 656 C ARG 418 8.370 125.926 82.605 657 O ARG 418 7.969 125.228 83.535 658 CB ARG 418 7.848 124.135 81.000 659 CG ARG 418 8.361 122.979 80.130 660 CD ARG 418 8.588 123.290 78.646 661 NE ARG 418 9.926 123.849 78.405 662 CZ ARG 418 11.023 123.101 78.282 663 NH1 ARG 418 10.910 121.793 78.039 664 NH2 ARG 418 12.216 123.689 78.184 665 N TYR 419 8.275 127.249 82.557 666 CA TYR 419 8.081 128.120 83.735 667 C TYR 419 7.189 127.499 84.806 668 O TYR 419 6.025 127.167 84.561 669 CB TYR 419 7.496 129.444 83.254 670 CG TYR 419 7.573 130.575 84.277 671 CD1 TYR 419 8.785 131.216 84.500 672 CD2 TYR 419 6.440 130.959 84.983 673 CE1 TYR 419 8.866 132.247 85.427 674 CE2 TYR 419 6.518 131.990 85.910 675 CZ TYR 419 7.731 132.632 86.128 676 OH TYR 419 7.808 133.659 87.041 677 N ASP 420 7.758 127.423 86.003 678 CA ASP 420 7.234 126.630 87.133 679 C ASP 420 7.448 125.166 86.770 680 O ASP 420 6.605 124.330 87.121 681 CB ASP 420 5.746 126.888 87.399 682 CG ASP 420 5.469 128.372 87.621 683 OD1 ASP 420 6.344 129.026 88.168 684 OD2 ASP 420 4.376 128.816 87.296 685 N SER 421 8.704 124.873 86.451 686 CA SER 421 9.095 123.693 85.647 687 C SER 421 8.423 122.418 86.083 688 O SER 421 7.707 121.785 85.302 689 CB SER 421 10.604 123.498 85.670 690 OG SER 421 11.207 124.686 85.178 691 N ASP 422 8.690 121.997 87.300 692 CA ASP 422 7.950 120.854 87.819 693 C ASP 422 7.255 121.244 89.114 694 O ASP 422 7.892 121.677 90.084 695 CB ASP 422 8.866 119.646 87.984 696 CG ASP 422 9.388 119.186 86.619 697 OD1 ASP 422 8.724 118.374 85.988 698 OD2 ASP 422 10.481 119.610 86.264 699 N LYS 423 5.937 121.185 89.078 700 CA LYS 423 5.134 121.525 90.250 701 C LYS 423 4.311 120.317 90.688 702 O LYS 423 3.336 119.931 90.035 703 CB LYS 423 4.231 122.699 89.902 704 CG LYS 423 3.490 123.199 91.135 705 CD LYS 423 2.684 124.456 90.834 706 CE LYS 423 2.021 124.987 92.099 707 NZ LYS 423 1.151 123.965 92.701 708 N ASP 424 4.713 119.767 91.823 709 CA ASP 424 4.130 118.535 92.376 710 C ASP 424 4.097 117.412 91.335 711 O ASP 424 3.035 117.070 90.802 712 CB ASP 424 2.724 118.845 92.887 713 CG ASP 424 2.173 117.669 93.686 714 OD1 ASP 424 2.971 117.013 94.343 715 OD2 ASP 424 0.963 117.499 93.693 716 N GLN 425 5.272 116.842 91.095 717 CA GLN 425 5.491 115.756 90.116 718 C GLN 425 4.794 116.022 88.771 719 O GLN 425 3.972 115.215 88.324 720 CB GLN 425 4.997 114.447 90.746 721 CG GLN 425 5.483 113.189 90.019 722 CD GLN 425 5.005 111.937 90.756 723 OE1 GLN 425 3.925 111.934 91.358 724 NE2 GLN 425 5.818 110.891 90.727 725 N ALA 426 5.219 117.075 88.087 726 CA ALA 426 4.524 117.493 86.854 727 C ALA 426 5.214 118.643 86.121 728 O ALA 426 5.323 119.756 86.648 729 CB ALA 426 3.106 117.930 87.211 730 N LEU 427 5.623 118.369 84.894 731 CA LEU 427 6.248 119.382 84.032 732 C LEU 427 5.190 120.344 83.471 733 O LEU 427 4.127 119.898 83.019 734 CB LEU 427 6.948 118.636 82.894 735 CG LEU 427 7.863 119.529 82.061 736 CD1 LEU 427 9.007 120.075 82.908 737 CD2 LEU 427 8.418 118.770 80.862 738 N THR 428 5.455 121.638 83.564 739 CA THR 428 4.541 122.667 83.037 740 C THR 428 4.317 122.508 81.536 741 O THR 428 5.252 122.328 80.747 742 CB THR 428 5.133 124.052 83.290 743 OG1 THR 428 5.309 124.232 84.684 744 CG2 THR 428 4.254 125.195 82.784 745 N GLU 429 3.054 122.559 81.154 746 CA GLU 429 2.660 122.538 79.740 747 C GLU 429 2.869 123.904 79.063 748 O GLU 429 1.913 124.681 78.958 749 CB GLU 429 1.173 122.175 79.737 750 CG GLU 429 0.543 122.076 78.352 751 CD GLU 429 −0.961 121.864 78.516 752 OE1 GLU 429 −1.597 121.489 77.542 753 OE2 GLU 429 −1.427 122.000 79.639 754 N ASP 430 4.096 124.214 78.654 755 CA ASP 430 4.326 125.458 77.891 756 C ASP 430 5.716 125.550 77.240 757 O ASP 430 6.727 125.705 77.929 758 CB ASP 430 4.124 126.660 78.812 759 CG ASP 430 3.722 127.873 77.979 760 OD1 ASP 430 3.331 127.667 76.837 761 OD2 ASP 430 3.821 128.982 78.488 762 N GLU 431 5.679 125.651 75.916 763 CA GLU 431 6.827 125.841 74.994 764 C GLU 431 8.199 125.325 75.435 765 O GLU 431 8.896 125.954 76.243 766 CB GLU 431 6.961 127.332 74.684 767 CG GLU 431 8.028 127.608 73.621 768 CD GLU 431 7.601 127.033 72.267 769 OE1 GLU 431 6.896 127.741 71.563 770 OE2 GLU 431 7.800 125.843 72.064 771 N GLN 432 8.630 124.263 74.776 772 CA GLN 432 10.001 123.766 74.936 773 C GLN 432 11.007 124.753 74.345 774 O GLN 432 10.772 125.362 73.295 775 CB GLN 432 10.154 122.382 74.300 776 CG GLN 432 9.574 122.279 72.889 777 CD GLN 432 8.193 121.619 72.929 778 OE1 GLN 432 7.183 122.278 73.206 779 NE2 GLN 432 8.186 120.311 72.743 780 N GLY 433 12.077 124.969 75.093 781 CA GLY 433 13.131 125.911 74.704 782 C GLY 433 12.638 127.354 74.787 783 O GLY 433 12.899 128.159 73.884 784 N LYS 434 11.921 127.670 75.851 785 CA LYS 434 11.337 129.008 75.977 786 C LYS 434 12.353 130.098 76.293 787 O LYS 434 13.489 129.826 76.704 788 CB LYS 434 10.238 128.991 77.024 789 CG LYS 434 10.625 128.200 78.263 790 CD LYS 434 9.548 128.404 79.312 791 CE LYS 434 8.172 128.322 78.669 792 NZ LYS 434 7.112 128.661 79.627 793 N SER 435 11.920 131.304 75.946 794 CA SER 435 12.619 132.607 76.061 795 C SER 435 13.623 132.778 77.204 796 O SER 435 13.385 133.556 78.139 797 CB SER 435 11.511 133.634 76.240 798 OG SER 435 10.702 133.195 77.328 799 N TYR 436 14.841 132.351 76.928 800 CA TYR 436 15.894 132.296 77.942 801 C TYR 436 16.373 133.679 78.416 802 O TYR 436 16.005 134.013 79.545 803 CB TYR 436 17.013 131.313 77.541 804 CG TYR 436 17.346 131.096 76.052 805 CD1 TYR 436 16.493 130.368 75.228 806 CD2 TYR 436 18.549 131.572 75.548 807 CE1 TYR 436 16.812 130.167 73.895 808 CE2 TYR 436 18.871 131.374 74.208 809 CZ TYR 436 17.999 130.674 73.383 810 OH TYR 436 18.348 130.423 72.078 811 N PRO 437 16.939 134.558 77.595 812 CA PRO 437 17.220 135.909 78.097 813 C PRO 437 16.010 136.851 78.042 814 O PRO 437 16.074 137.957 78.591 815 CB PRO 437 18.281 136.420 77.171 816 CG PRO 437 18.282 135.576 75.907 817 CD PRO 437 17.297 134.453 76.173 818 N LYS 438 14.886 136.356 77.550 819 CA LYS 438 13.818 137.231 77.084 820 C LYS 438 12.646 137.309 78.056 821 O LYS 438 11.964 138.340 78.112 822 CB LYS 438 13.388 136.645 75.742 823 CG LYS 438 12.327 137.451 75.007 824 CD LYS 438 12.041 136.807 73.651 825 CE LYS 438 10.998 137.583 72.856 826 NZ LYS 438 10.774 136.962 71.541 827 N LEU 439 12.452 136.280 78.859 828 CA LEU 439 11.358 136.355 79.825 829 C LEU 439 11.881 136.461 81.246 830 O LEU 439 11.232 137.093 82.087 831 CB LEU 439 10.464 135.129 79.694 832 CG LEU 439 9.205 135.256 80.549 833 CD1 LEU 439 8.425 136.519 80.190 834 CD2 LEU 439 8.316 134.026 80.415 835 N ILE 440 13.031 135.871 81.518 836 CA ILE 440 13.559 135.971 82.878 837 C ILE 440 14.624 137.055 83.000 838 O ILE 440 15.331 137.372 82.034 839 CB ILE 440 14.070 134.615 83.356 840 CG1 ILE 440 15.163 134.068 82.455 841 CG2 ILE 440 12.915 133.622 83.456 842 CD1 ILE 440 15.642 132.701 82.931 843 N SER 441 14.483 137.770 84.107 844 CA SER 441 15.381 138.832 84.598 845 C SER 441 15.417 140.051 83.687 846 O SER 441 16.350 140.853 83.800 847 CB SER 441 16.803 138.308 84.780 848 OG SER 441 17.452 138.283 83.516 849 N GLU 442 14.297 140.321 83.040 850 CA GLU 442 14.190 141.386 82.032 851 C GLU 442 15.479 141.595 81.228 852 O GLU 442 16.187 142.585 81.450 853 CB GLU 442 13.831 142.676 82.768 854 CG GLU 442 13.477 143.820 81.820 855 CD GLU 442 13.302 145.108 82.618 856 OE1 GLU 442 13.235 146.158 81.995 857 OE2 GLU 442 13.110 145.003 83.822 858 N GLY 443 15.827 140.640 80.379 859 CA GLY 443 16.932 140.860 79.440 860 C GLY 443 18.317 140.429 79.925 861 O GLY 443 19.165 141.274 80.237 862 N PHE 444 18.558 139.128 79.920 863 CA PHE 444 19.927 138.619 80.102 864 C PHE 444 20.770 138.951 78.872 865 O PHE 444 20.244 139.030 77.757 866 CB PHE 444 19.904 137.102 80.271 867 CG PHE 444 19.702 136.592 81.688 868 CD1 PHE 444 20.458 137.116 82.728 869 CD2 PHE 444 18.787 135.577 81.935 870 CE1 PHE 444 20.286 136.641 84.019 871 CE2 PHE 444 18.615 135.101 83.228 872 CZ PHE 444 19.363 135.635 84.270 873 N PRO 445 22.057 139.179 79.081 874 CA PRO 445 22.979 139.324 77.952 875 C PRO 445 23.018 138.046 77.114 876 O PRO 445 22.963 136.935 77.649 877 CB PRO 445 24.310 139.633 78.565 878 CG PRO 445 24.193 139.561 80.081 879 CD PRO 445 22.740 139.225 80.377 880 N GLY 446 23.187 138.223 75.813 881 CA GLY 446 23.173 137.095 74.866 882 C GLY 446 24.482 136.306 74.818 883 O GLY 446 24.514 135.168 74.333 884 N ILE 447 25.559 136.928 75.267 885 CA ILE 447 26.859 136.246 75.336 886 C ILE 447 26.895 135.074 76.347 887 O ILE 447 27.365 134.005 75.937 888 CB ILE 447 27.941 137.278 75.654 889 CG1 ILE 447 27.879 138.438 74.668 890 CG2 ILE 447 29.326 136.639 75.627 891 CD1 ILE 447 28.932 139.492 74.990 892 N PRO 448 26.507 135.233 77.614 893 CA PRO 448 26.389 134.052 78.481 894 C PRO 448 25.324 133.079 77.996 895 O PRO 448 24.121 133.364 78.024 896 CB PRO 448 26.022 134.564 79.835 897 CG PRO 448 25.830 136.065 79.775 898 CD PRO 448 26.133 136.458 78.343 899 N SER 449 25.787 131.905 77.615 900 CA SER 449 24.880 130.840 77.191 901 C SER 449 24.521 129.850 78.316 902 O SER 449 23.333 129.797 78.654 903 CB SER 449 25.463 130.152 75.960 904 OG SER 449 24.809 128.902 75.822 905 N PRO 450 25.440 129.059 78.868 906 CA PRO 450 25.032 128.115 79.910 907 C PRO 450 24.914 128.779 81.277 908 O PRO 450 25.823 129.488 81.724 909 CB PRO 450 26.107 127.074 79.937 910 CG PRO 450 27.316 127.604 79.182 911 CD PRO 450 26.881 128.933 78.588 912 N LEU 451 23.781 128.565 81.922 913 CA LEU 451 23.648 129.003 83.319 914 C LEU 451 24.256 127.956 84.248 915 O LEU 451 23.834 126.790 84.273 916 CB LEU 451 22.186 129.258 83.681 917 CG LEU 451 21.742 130.697 83.398 918 CD1 LEU 451 22.753 131.688 83.969 919 CD2 LEU 451 21.500 130.982 81.917 920 N ASP 452 25.199 128.401 85.063 921 CA ASP 452 25.946 127.463 85.905 922 C ASP 452 25.188 127.169 87.188 923 O ASP 452 24.663 126.061 87.351 924 CB ASP 452 27.310 128.052 86.236 925 CG ASP 452 28.089 128.391 84.973 926 OD1 ASP 452 28.088 127.583 84.054 927 OD2 ASP 452 28.652 129.481 84.938 928 N THR 453 25.056 128.163 88.045 929 CA THR 453 24.306 127.963 89.289 930 C THR 453 23.238 129.035 89.481 931 O THR 453 23.511 130.231 89.383 932 CB THR 453 25.292 127.983 90.451 933 OG1 THR 453 26.204 126.907 90.268 934 CG2 THR 453 24.601 127.789 91.794 935 N ALA 454 22.011 128.606 89.706 936 CA ALA 454 20.946 129.565 90.004 937 C ALA 454 20.296 129.215 91.336 938 O ALA 454 19.862 128.074 91.530 939 CB ALA 454 19.921 129.529 88.878 940 N PHE 455 20.278 130.161 92.259 941 CA PHE 455 19.649 129.885 93.558 942 C PHE 455 18.694 131.004 93.975 943 O PHE 455 18.970 132.193 93.779 944 CB PHE 455 20.727 129.656 94.623 945 CG PHE 455 21.082 130.843 95.518 946 CD1 PHE 455 21.951 131.829 95.070 947 CD2 PHE 455 20.539 130.926 96.794 948 CE1 PHE 455 22.265 132.904 95.894 949 CE2 PHE 455 20.851 132.000 97.615 950 CZ PHE 455 21.712 132.990 97.166 951 N TYR 456 17.556 130.617 94.519 952 CA TYR 456 16.598 131.614 94.999 953 C TYR 456 16.882 131.976 96.453 954 O TYR 456 16.842 131.119 97.342 955 CB TYR 456 15.184 131.057 94.871 956 CG TYR 456 14.130 131.904 95.582 957 CD1 TYR 456 13.893 133.210 95.172 958 CD2 TYR 456 13.415 131.366 96.643 959 CE1 TYR 456 12.951 133.984 95.836 960 CE2 TYR 456 12.471 132.139 97.307 961 CZ TYR 456 12.247 133.449 96.906 962 OH TYR 456 11.401 134.254 97.636 963 N ASP 457 17.195 133.235 96.698 964 CA ASP 457 17.354 133.665 98.082 965 C ASP 457 16.010 134.116 98.642 966 O ASP 457 15.526 135.223 98.370 967 CB ASP 457 18.369 134.792 98.187 968 CG ASP 457 18.559 135.090 99.666 969 OD1 ASP 457 18.176 136.185 100.045 970 OD2 ASP 457 18.626 134.114 100.401 971 N ARG 458 15.560 133.341 99.616 972 CA ARG 458 14.245 133.532 100.234 973 C ARG 458 14.157 134.783 101.106 974 O ARG 458 13.186 135.532 100.963 975 CB ARG 458 13.991 132.293 101.090 976 CG ARG 458 12.746 132.407 101.962 977 CD ARG 458 12.585 131.153 102.816 978 NE ARG 458 11.491 131.293 103.789 979 CZ ARG 458 11.437 130.573 104.912 980 NH1 ARG 458 12.384 129.668 105.168 981 NH2 ARG 458 10.435 130.754 105.776 982 N ARG 459 15.282 135.177 101.684 983 CA ARG 459 15.293 136.315 102.614 984 C ARG 459 15.329 137.684 101.931 985 O ARG 459 15.214 138.707 102.613 986 CB ARG 459 16.517 136.177 103.512 987 CG ARG 459 16.435 134.915 104.363 988 CD ARG 459 15.223 134.959 105.287 989 NE ARG 459 15.133 133.739 106.103 990 CZ ARG 459 13.972 133.225 106.513 991 NH1 ARG 459 13.959 132.165 107.321 992 NH2 ARG 459 12.825 133.816 106.166 993 N GLN 460 15.529 137.707 100.622 994 CA GLN 460 15.506 138.966 99.877 995 C GLN 460 14.505 138.899 98.726 996 O GLN 460 14.343 139.886 98.001 997 CB GLN 460 16.898 139.222 99.301 998 CG GLN 460 17.987 139.354 100.365 999 CD GLN 460 17.804 140.623 101.194 1000 OE1 GLN 460 17.613 141.715 100.649 1001 NE2 GLN 460 17.943 140.472 102.501 1002 N LYS 461 13.824 137.761 98.610 1003 CA LYS 461 12.999 137.369 97.438 1004 C LYS 461 13.679 137.707 96.102 1005 O LYS 461 13.074 138.238 95.155 1006 CB LYS 461 11.564 137.902 97.554 1007 CG LYS 461 11.430 139.420 97.629 1008 CD LYS 461 10.029 139.840 98.055 1009 CE LYS 461 9.905 141.358 98.096 1010 NZ LYS 461 10.903 141.941 99.006 1011 N LEU 462 14.923 137.257 96.018 1012 CA LEU 462 15.797 137.525 94.866 1013 C LEU 462 16.533 136.263 94.443 1014 O LEU 462 17.305 135.685 95.218 1015 CB LEU 462 16.871 138.550 95.236 1016 CG LEU 462 16.333 139.927 95.607 1017 CD1 LEU 462 17.464 140.845 96.051 1018 CD2 LEU 462 15.560 140.559 94.461 1019 N ILE 463 16.349 135.875 93.198 1020 CA ILE 463 17.083 134.731 92.659 1021 C ILE 463 18.374 135.213 91.996 1022 O ILE 463 18.377 136.145 91.182 1023 CB ILE 463 16.168 133.979 91.696 1024 CG1 ILE 463 16.904 132.948 90.854 1025 CG2 ILE 463 15.400 134.939 90.808 1026 CD1 ILE 463 15.960 132.352 89.818 1027 N TYR 464 19.477 134.645 92.446 1028 CA TYR 464 20.792 135.039 91.941 1029 C TYR 464 21.311 134.041 90.911 1030 O TYR 464 21.390 132.827 91.150 1031 CB TYR 464 21.768 135.166 93.106 1032 CG TYR 464 21.420 136.275 94.100 1033 CD1 TYR 464 20.729 135.977 95.270 1034 CD2 TYR 464 21.810 137.581 93.839 1035 CE1 TYR 464 20.414 136.988 96.167 1036 CE2 TYR 464 21.496 138.593 94.738 1037 CZ TYR 464 20.796 138.294 95.897 1038 OH TYR 464 20.455 139.300 96.775 1039 N PHE 465 21.616 134.586 89.747 1040 CA PHE 465 22.168 133.807 88.636 1041 C PHE 465 23.677 133.935 88.541 1042 O PHE 465 24.231 135.020 88.330 1043 CB PHE 465 21.550 134.271 87.327 1044 CG PHE 465 20.191 133.655 87.042 1045 CD1 PHE 465 20.125 132.438 86.380 1046 CD2 PHE 465 19.026 134.295 87.440 1047 CE1 PHE 465 18.892 131.861 86.106 1048 CE2 PHE 465 17.795 133.718 87.165 1049 CZ PHE 465 17.725 132.503 86.498 1050 N PHE 466 24.320 132.793 88.652 1051 CA PHE 466 25.770 132.708 88.602 1052 C PHE 466 26.265 132.161 87.263 1053 O PHE 466 26.094 130.981 86.916 1054 CB PHE 466 26.214 131.791 89.733 1055 CG PHE 466 26.041 132.326 91.150 1056 CD1 PHE 466 27.075 133.039 91.739 1057 CD2 PHE 466 24.874 132.080 91.862 1058 CE1 PHE 466 26.935 133.527 93.031 1059 CE2 PHE 466 24.732 132.570 93.152 1060 CZ PHE 466 25.761 133.296 93.736 1061 N LYS 467 26.928 133.041 86.534 1062 CA LYS 467 27.579 132.661 85.279 1063 C LYS 467 29.055 133.032 85.401 1064 O LYS 467 29.410 134.211 85.281 1065 CB LYS 467 26.904 133.405 84.127 1066 CG LYS 467 27.104 132.737 82.763 1067 CD LYS 467 28.510 132.898 82.188 1068 CE LYS 467 28.882 134.367 82.017 1069 NZ LYS 467 30.241 134.511 81.471 1070 N GLU 468 29.901 132.013 85.418 1071 CA GLU 468 31.321 132.159 85.782 1072 C GLU 468 31.503 133.103 86.967 1073 O GLU 468 31.038 132.802 88.072 1074 CB GLU 468 32.117 132.645 84.577 1075 CG GLU 468 32.111 131.607 83.457 1076 CD GLU 468 32.693 130.287 83.961 1077 OE1 GLU 468 31.906 129.436 84.356 1078 OE2 GLU 468 33.909 130.182 84.003 1079 N SER 469 32.110 134.252 86.715 1080 CA SER 469 32.377 135.226 87.779 1081 C SER 469 31.287 136.282 87.946 1082 O SER 469 31.485 137.215 88.733 1083 CB SER 469 33.684 135.945 87.472 1084 OG SER 469 33.462 136.771 86.338 1085 N LEU 470 30.201 136.189 87.196 1086 CA LEU 470 29.145 137.209 87.261 1087 C LEU 470 27.913 136.738 88.035 1088 O LEU 470 27.389 135.641 87.805 1089 CB LEU 470 28.723 137.552 85.835 1090 CG LEU 470 29.906 137.970 84.967 1091 CD1 LEU 470 29.480 138.124 83.512 1092 CD2 LEU 470 30.565 139.249 85.474 1093 N VAL 471 27.473 137.587 88.949 1094 CA VAL 471 26.247 137.366 89.729 1095 C VAL 471 25.156 138.357 89.331 1096 O VAL 471 25.285 139.569 89.550 1097 CB VAL 471 26.559 137.573 91.207 1098 CG1 VAL 471 25.331 137.328 92.079 1099 CG2 VAL 471 27.707 136.687 91.658 1100 N PHE 472 24.089 137.822 88.766 1101 CA PHE 472 22.918 138.617 88.385 1102 C PHE 472 21.821 138.533 89.445 1103 O PHE 472 21.343 137.439 89.768 1104 CB PHE 472 22.380 138.099 87.054 1105 CG PHE 472 23.367 138.172 85.890 1106 CD1 PHE 472 23.575 139.381 85.238 1107 CD2 PHE 472 24.049 137.035 85.478 1108 CE1 PHE 472 24.472 139.453 84.180 1109 CE2 PHE 472 24.947 137.107 84.420 1110 CZ PHE 472 25.158 138.317 83.773 1111 N ALA 473 21.431 139.676 89.981 1112 CA ALA 473 20.366 139.698 91.001 1113 C ALA 473 19.003 139.971 90.379 1114 O ALA 473 18.744 141.079 89.905 1115 CB ALA 473 20.670 140.780 92.027 1116 N PHE 474 18.126 138.985 90.431 1117 CA PHE 474 16.811 139.100 89.788 1118 C PHE 474 15.650 139.026 90.794 1119 O PHE 474 15.505 138.067 91.560 1120 CB PHE 474 16.819 137.996 88.719 1121 CG PHE 474 15.542 137.320 88.209 1122 CD1 PHE 474 15.674 136.064 87.632 1123 CD2 PHE 474 14.289 137.914 88.266 1124 CE1 PHE 474 14.560 135.379 87.167 1125 CE2 PHE 474 13.174 137.227 87.810 1126 CZ PHE 474 13.306 135.958 87.267 1127 N ASP 475 14.836 140.071 90.787 1128 CA ASP 475 13.612 140.102 91.599 1129 C ASP 475 12.571 139.188 90.968 1130 O ASP 475 12.008 139.525 89.916 1131 CB ASP 475 13.081 141.534 91.652 1132 CG ASP 475 11.740 141.583 92.383 1133 OD1 ASP 475 11.744 141.608 93.604 1134 OD2 ASP 475 10.726 141.412 91.712 1135 N VAL 476 12.155 138.182 91.721 1136 CA VAL 476 11.336 137.103 91.149 1137 C VAL 476 9.904 137.517 90.830 1138 O VAL 476 9.419 137.225 89.731 1139 CB VAL 476 11.271 135.987 92.181 1140 CG1 VAL 476 10.720 134.711 91.556 1141 CG2 VAL 476 12.640 135.728 92.783 1142 N ASN 477 9.383 138.437 91.621 1143 CA ASN 477 7.965 138.807 91.548 1144 C ASN 477 7.622 139.608 90.294 1145 O ASN 477 6.593 139.355 89.657 1146 CB ASN 477 7.670 139.679 92.766 1147 CG ASN 477 8.325 139.091 94.015 1148 OD1 ASN 477 8.081 137.935 94.385 1149 ND2 ASN 477 9.179 139.888 94.635 1150 N ARG 478 8.502 140.518 89.915 1151 CA ARG 478 8.250 141.350 88.734 1152 C ARG 478 9.104 140.933 87.543 1153 O ARG 478 8.986 141.514 86.457 1154 CB ARG 478 8.523 142.798 89.105 1155 CG ARG 478 7.627 143.224 90.259 1156 CD ARG 478 7.890 144.668 90.665 1157 NE ARG 478 7.067 145.048 91.821 1158 CZ ARG 478 7.539 145.105 93.068 1159 NH1 ARG 478 8.820 144.821 93.315 1160 NH2 ARG 478 6.729 145.457 94.071 1161 N ASN 479 9.972 139.961 87.783 1162 CA ASN 479 10.829 139.353 86.762 1163 C ASN 479 11.825 140.352 86.168 1164 O ASN 479 12.074 140.357 84.955 1165 CB ASN 479 9.930 138.757 85.686 1166 CG ASN 479 10.577 137.513 85.101 1167 OD1 ASN 479 11.808 137.407 85.044 1168 ND2 ASN 479 9.740 136.552 84.755 1169 N ARG 480 12.505 141.072 87.047 1170 CA ARG 480 13.450 142.119 86.616 1171 C ARG 480 14.816 141.960 87.281 1172 O ARG 480 14.878 141.751 88.499 1173 CB ARG 480 12.913 143.479 87.049 1174 CG ARG 480 11.497 143.766 86.567 1175 CD ARG 480 11.021 145.086 87.155 1176 NE ARG 480 11.251 145.087 88.609 1177 CZ ARG 480 10.993 146.128 89.403 1178 NH1 ARG 480 10.435 147.231 88.898 1179 NH2 ARG 480 11.253 146.049 90.710 1180 N VAL 481 15.889 142.083 86.516 1181 CA VAL 481 17.221 142.113 87.135 1182 C VAL 481 17.421 143.516 87.712 1183 O VAL 481 16.903 144.505 87.175 1184 CB VAL 481 18.308 141.750 86.118 1185 CG1 VAL 481 18.529 142.864 85.100 1186 CG2 VAL 481 19.624 141.371 86.796 1187 N LEU 482 18.060 143.580 88.863 1188 CA LEU 482 18.196 144.840 89.602 1189 C LEU 482 19.268 145.749 89.017 1190 O LEU 482 20.444 145.670 89.394 1191 CB LEU 482 18.539 144.507 91.047 1192 CG LEU 482 17.438 143.678 91.698 1193 CD1 LEU 482 17.841 143.269 93.109 1194 CD2 LEU 482 16.111 144.432 91.716 1195 N ASN 483 18.809 146.668 88.178 1196 CA ASN 483 19.661 147.650 87.483 1197 C ASN 483 20.956 147.008 87.004 1198 O ASN 483 22.038 147.308 87.526 1199 CB ASN 483 19.948 148.809 88.434 1200 CG ASN 483 18.641 149.512 88.800 1201 OD1 ASN 483 18.000 149.187 89.807 1202 ND2 ASN 483 18.264 150.470 87.973 1203 N SER 484 20.799 146.129 86.020 1204 CA SER 484 21.809 145.194 85.454 1205 C SER 484 22.448 144.177 86.433 1206 O SER 484 22.346 142.976 86.153 1207 CB SER 484 22.892 145.945 84.663 1208 OG SER 484 23.749 146.693 85.516 1209 N TYR 485 22.928 144.622 87.588 1210 CA TYR 485 23.686 143.829 88.585 1211 C TYR 485 24.365 142.571 88.028 1212 O TYR 485 23.779 141.489 88.138 1213 CB TYR 485 22.693 143.429 89.676 1214 CG TYR 485 23.268 143.028 91.038 1215 CD1 TYR 485 24.063 141.896 91.177 1216 CD2 TYR 485 22.966 143.799 92.153 1217 CE1 TYR 485 24.558 141.537 92.424 1218 CE2 TYR 485 23.458 143.443 93.402 1219 CZ TYR 485 24.252 142.312 93.533 1220 OH TYR 485 24.712 141.936 94.775 1221 N PRO 486 25.459 142.727 87.289 1222 CA PRO 486 26.309 141.605 86.864 1223 C PRO 486 27.534 141.407 87.766 1224 O PRO 486 28.555 140.897 87.291 1225 CB PRO 486 26.791 142.065 85.525 1226 CG PRO 486 26.781 143.590 85.537 1227 CD PRO 486 26.046 143.985 86.811 1228 N LYS 487 27.426 141.752 89.039 1229 CA LYS 487 28.615 141.994 89.871 1230 C LYS 487 29.491 140.765 90.095 1231 O LYS 487 29.034 139.622 90.032 1232 CB LYS 487 28.158 142.583 91.196 1233 CG LYS 487 27.456 143.913 90.947 1234 CD LYS 487 27.000 144.573 92.243 1235 CE LYS 487 26.270 145.881 91.960 1236 NZ LYS 487 25.790 146.497 93.206 1237 N ARG 488 30.772 141.036 90.276 1238 CA ARG 488 31.785 139.991 90.469 1239 C ARG 488 31.458 139.127 91.682 1240 O ARG 488 31.213 139.660 92.775 1241 CB ARG 488 33.108 140.711 90.711 1242 CG ARG 488 34.319 139.787 90.757 1243 CD ARG 488 35.535 140.550 91.270 1244 NE ARG 488 35.658 141.840 90.574 1245 CZ ARG 488 36.656 142.146 89.742 1246 NH1 ARG 488 36.668 143.329 89.123 1247 NH2 ARG 488 37.626 141.259 89.505 1248 N ILE 489 31.610 137.819 91.526 1249 CA ILE 489 31.345 136.867 92.614 1250 C ILE 489 32.141 137.164 93.878 1251 O ILE 489 31.512 137.388 94.915 1252 CB ILE 489 31.687 135.448 92.161 1253 CG1 ILE 489 30.687 134.912 91.153 1254 CG2 ILE 489 31.768 134.494 93.343 1255 CD1 ILE 489 30.849 133.405 91.018 1256 N THR 490 33.397 137.550 93.728 1257 CA THR 490 34.239 137.787 94.905 1258 C THR 490 33.947 139.122 95.610 1259 O THR 490 34.246 139.244 96.804 1260 CB THR 490 35.693 137.720 94.443 1261 OG1 THR 490 35.895 136.441 93.857 1262 CG2 THR 490 36.684 137.868 95.592 1263 N GLU 491 33.196 140.016 94.980 1264 CA GLU 491 32.889 141.288 95.642 1265 C GLU 491 31.476 141.290 96.231 1266 O GLU 491 31.127 142.225 96.961 1267 CB GLU 491 33.055 142.459 94.670 1268 CG GLU 491 31.898 142.575 93.684 1269 CD GLU 491 32.104 143.744 92.727 1270 OE1 GLU 491 31.666 143.614 91.589 1271 OE2 GLU 491 32.778 144.689 93.106 1272 N VAL 492 30.663 140.297 95.898 1273 CA VAL 492 29.326 140.223 96.498 1274 C VAL 492 29.215 139.006 97.410 1275 O VAL 492 28.291 138.898 98.220 1276 CB VAL 492 28.254 140.211 95.409 1277 CG1 VAL 492 28.130 141.579 94.752 1278 CG2 VAL 492 28.495 139.133 94.360 1279 N PHE 493 30.155 138.094 97.246 1280 CA PHE 493 30.317 136.939 98.132 1281 C PHE 493 31.812 136.673 98.325 1282 O PHE 493 32.411 135.866 97.598 1283 CB PHE 493 29.662 135.709 97.502 1284 CG PHE 493 28.154 135.804 97.271 1285 CD1 PHE 493 27.281 135.676 98.341 1286 CD2 PHE 493 27.656 136.016 95.991 1287 CE1 PHE 493 25.910 135.769 98.135 1288 CE2 PHE 493 26.287 136.111 95.786 1289 CZ PHE 493 25.412 135.988 96.857 1290 N PRO 494 32.411 137.388 99.267 1291 CA PRO 494 33.867 137.367 99.413 1292 C PRO 494 34.408 135.984 99.751 1293 O PRO 494 33.874 135.248 100.590 1294 CB PRO 494 34.184 138.368 100.478 1295 CG PRO 494 32.897 139.020 100.949 1296 CD PRO 494 31.776 138.348 100.174 1297 N ALA 495 35.416 135.624 98.971 1298 CA ALA 495 36.163 134.366 99.105 1299 C ALA 495 35.350 133.089 98.881 1300 O ALA 495 35.592 132.086 99.563 1301 CB ALA 495 36.829 134.318 100.478 1302 N VAL 496 34.428 133.106 97.931 1303 CA VAL 496 33.776 131.852 97.528 1304 C VAL 496 34.274 131.378 96.162 1305 O VAL 496 33.688 130.452 95.586 1306 CB VAL 496 32.257 132.003 97.498 1307 CG1 VAL 496 31.708 132.382 98.865 1308 CG2 VAL 496 31.810 133.003 96.445 1309 N ILE 497 35.322 132.022 95.660 1310 CA ILE 497 35.932 131.700 94.352 1311 C ILE 497 35.025 132.075 93.173 1312 O ILE 497 33.850 131.696 93.111 1313 CB ILE 497 36.347 130.221 94.343 1314 CG1 ILE 497 37.535 130.003 95.273 1315 CG2 ILE 497 36.670 129.694 92.950 1316 CD1 ILE 497 38.035 128.564 95.204 1317 N PRO 498 35.589 132.838 92.241 1318 CA PRO 498 34.854 133.407 91.093 1319 C PRO 498 34.414 132.427 89.982 1320 O PRO 498 34.080 132.879 88.884 1321 CB PRO 498 35.775 134.440 90.518 1322 CG PRO 498 37.128 134.351 91.204 1323 CD PRO 498 36.981 133.297 92.287 1324 N GLN 499 34.491 131.128 90.213 1325 CA GLN 499 33.956 130.160 89.249 1326 C GLN 499 33.034 129.187 89.981 1327 O GLN 499 33.422 128.095 90.411 1328 CB GLN 499 35.084 129.458 88.504 1329 CG GLN 499 36.179 128.929 89.421 1330 CD GLN 499 37.200 128.189 88.569 1331 OE1 GLN 499 38.398 128.164 88.875 1332 NE2 GLN 499 36.705 127.607 87.491 1333 N ASN 500 31.781 129.600 90.023 1334 CA ASN 500 30.742 128.989 90.857 1335 C ASN 500 30.364 127.546 90.515 1336 O ASN 500 30.345 127.109 89.357 1337 CB ASN 500 29.486 129.857 90.741 1338 CG ASN 500 28.833 129.735 89.359 1339 OD1 ASN 500 28.029 128.827 89.121 1340 ND2 ASN 500 29.108 130.682 88.481 1341 N HIS 501 30.139 126.809 91.588 1342 CA HIS 501 29.462 125.512 91.553 1343 C HIS 501 28.194 125.670 92.411 1344 O HIS 501 27.942 126.801 92.844 1345 CB HIS 501 30.440 124.455 92.068 1346 CG HIS 501 31.434 123.955 91.040 1347 ND1 HIS 501 31.312 122.836 90.299 1348 CD2 HIS 501 32.622 124.546 90.681 1349 CE1 HIS 501 32.392 122.713 89.502 1350 NE2 HIS 501 33.200 123.771 89.738 1351 N PRO 502 27.380 124.632 92.602 1352 CA PRO 502 26.060 124.787 93.237 1353 C PRO 502 26.052 125.515 94.575 1354 O PRO 502 27.071 125.670 95.262 1355 CB PRO 502 25.510 123.414 93.405 1356 CG PRO 502 26.428 122.439 92.710 1357 CD PRO 502 27.543 123.267 92.104 1358 N PHE 503 24.842 125.897 94.936 1359 CA PHE 503 24.611 126.795 96.061 1360 C PHE 503 23.329 126.400 96.795 1361 O PHE 503 22.230 126.810 96.405 1362 CB PHE 503 24.464 128.175 95.434 1363 CG PHE 503 24.713 129.369 96.338 1364 CD1 PHE 503 24.000 129.530 97.515 1365 CD2 PHE 503 25.659 130.314 95.962 1366 CE1 PHE 503 24.237 130.633 98.319 1367 CE2 PHE 503 25.897 131.418 96.768 1368 CZ PHE 503 25.186 131.578 97.947 1369 N ARG 504 23.486 125.630 97.857 1370 CA ARG 504 22.334 125.146 98.634 1371 C ARG 504 21.964 126.124 99.764 1372 O ARG 504 22.844 126.703 100.410 1373 CB ARG 504 22.704 123.761 99.167 1374 CG ARG 504 21.543 123.045 99.845 1375 CD ARG 504 20.348 122.845 98.915 1376 NE ARG 504 19.188 122.364 99.681 1377 CZ ARG 504 18.049 123.051 99.807 1378 NH1 ARG 504 17.849 124.148 99.074 1379 NH2 ARG 504 17.069 122.582 100.580 1380 N ASN 505 20.669 126.272 100.005 1381 CA ASN 505 20.113 127.221 100.994 1382 C ASN 505 19.846 126.670 102.405 1383 O ASN 505 19.156 127.354 103.176 1384 CB ASN 505 18.755 127.692 100.486 1385 CG ASN 505 18.825 128.596 99.261 1386 OD1 ASN 505 19.278 128.205 98.179 1387 ND2 ASN 505 18.302 129.794 99.445 1388 N ILE 506 20.322 125.475 102.730 1389 CA ILE 506 19.920 124.780 103.979 1390 C ILE 506 20.039 125.638 105.244 1391 O ILE 506 21.093 126.206 105.549 1392 CB ILE 506 20.760 123.512 104.126 1393 CG1 ILE 506 20.371 122.482 103.079 1394 CG2 ILE 506 20.649 122.896 105.517 1395 CD1 ILE 506 21.221 121.225 103.214 1396 N ASP 507 18.895 125.799 105.903 1397 CA ASP 507 18.754 126.561 107.163 1398 C ASP 507 19.199 128.017 107.042 1399 O ASP 507 19.864 128.535 107.948 1400 CB ASP 507 19.560 125.882 108.268 1401 CG ASP 507 19.002 124.496 108.571 1402 OD1 ASP 507 17.792 124.338 108.477 1403 OD2 ASP 507 19.799 123.602 108.815 1404 N SER 508 18.867 128.635 105.915 1405 CA SER 508 19.215 130.031 105.600 1406 C SER 508 20.727 130.270 105.566 1407 O SER 508 21.196 131.379 105.853 1408 CB SER 508 18.564 130.957 106.622 1409 OG SER 508 17.169 130.693 106.606 1410 N ALA 509 21.471 129.241 105.199 1411 CA ALA 509 22.919 129.356 105.083 1412 C ALA 509 23.307 129.207 103.626 1413 O ALA 509 22.637 128.515 102.849 1414 CB ALA 509 23.582 128.276 105.925 1415 N TYR 510 24.372 129.889 103.256 1416 CA TYR 510 24.788 129.905 101.857 1417 C TYR 510 25.880 128.880 101.597 1418 O TYR 510 27.073 129.153 101.778 1419 CB TYR 510 25.278 131.305 101.498 1420 CG TYR 510 24.194 132.372 101.304 1421 CD1 TYR 510 24.576 133.681 101.045 1422 CD2 TYR 510 22.844 132.039 101.338 1423 CE1 TYR 510 23.613 134.664 100.858 1424 CE2 TYR 510 21.879 133.021 101.152 1425 CZ TYR 510 22.268 134.332 100.918 1426 OH TYR 510 21.316 135.318 100.780 1427 N TYR 511 25.474 127.720 101.116 1428 CA TYR 511 26.433 126.653 100.810 1429 C TYR 511 26.880 126.713 99.357 1430 O TYR 511 26.449 125.904 98.521 1431 CB TYR 511 25.801 125.298 101.096 1432 CG TYR 511 25.432 125.095 102.560 1433 CD1 TYR 511 24.110 125.203 102.964 1434 CD2 TYR 511 26.423 124.793 103.488 1435 CE1 TYR 511 23.777 125.032 104.299 1436 CE2 TYR 511 26.090 124.617 104.824 1437 CZ TYR 511 24.767 124.744 105.226 1438 OH TYR 511 24.434 124.618 106.556 1439 N SER 512 27.734 127.682 99.078 1440 CA SER 512 28.312 127.841 97.745 1441 C SER 512 29.564 126.996 97.646 1442 O SER 512 30.486 127.148 98.453 1443 CB SER 512 28.702 129.298 97.542 1444 OG SER 512 29.376 129.382 96.293 1445 N TYR 513 29.601 126.079 96.704 1446 CA TYR 513 30.812 125.280 96.646 1447 C TYR 513 31.561 125.346 95.328 1448 O TYR 513 31.227 126.128 94.428 1449 CB TYR 513 30.548 123.863 97.121 1450 CG TYR 513 29.579 122.904 96.442 1451 CD1 TYR 513 29.829 122.425 95.164 1452 CD2 TYR 513 28.470 122.462 97.151 1453 CE1 TYR 513 28.992 121.464 94.614 1454 CE2 TYR 513 27.627 121.510 96.597 1455 CZ TYR 513 27.904 120.998 95.340 1456 OH TYR 513 27.237 119.878 94.909 1457 N ALA 514 32.722 124.717 95.363 1458 CA ALA 514 33.615 124.668 94.206 1459 C ALA 514 34.350 123.334 94.155 1460 O ALA 514 35.148 123.017 95.050 1461 CB ALA 514 34.619 125.813 94.298 1462 N TYR 515 34.223 122.685 93.008 1463 CA TYR 515 34.776 121.346 92.747 1464 C TYR 515 34.484 120.364 93.874 1465 O TYR 515 33.361 119.861 93.969 1466 CB TYR 515 36.278 121.456 92.500 1467 CG TYR 515 36.633 122.301 91.280 1468 CD1 TYR 515 36.375 121.814 90.005 1469 CD2 TYR 515 37.203 123.559 91.442 1470 CE1 TYR 515 36.690 122.581 88.891 1471 CE2 TYR 515 37.516 124.328 90.329 1472 CZ TYR 515 37.259 123.837 89.057 1473 OH TYR 515 37.577 124.593 87.952 1474 N ASN 516 35.481 120.092 94.701 1475 CA ASN 516 35.334 119.132 95.803 1476 C ASN 516 35.174 119.765 97.197 1477 O ASN 516 34.841 119.051 98.150 1478 CB ASN 516 36.543 118.200 95.768 1479 CG ASN 516 36.519 117.393 94.469 1480 OD1 ASN 516 35.596 116.600 94.245 1481 ND2 ASN 516 37.541 117.569 93.646 1482 N SER 517 35.302 121.080 97.315 1483 CA SER 517 35.149 121.733 98.635 1484 C SER 517 33.861 122.557 98.730 1485 O SER 517 33.322 122.957 97.691 1486 CB SER 517 36.345 122.648 98.855 1487 OG SER 517 37.515 121.844 98.776 1488 N ILE 518 33.315 122.691 99.934 1489 CA ILE 518 32.135 123.557 100.151 1490 C ILE 518 32.463 124.730 101.083 1491 O ILE 518 33.282 124.607 102.005 1492 CB ILE 518 30.965 122.742 100.727 1493 CG1 ILE 518 29.616 123.405 100.472 1494 CG2 ILE 518 31.098 122.531 102.227 1495 CD1 ILE 518 28.483 122.580 101.070 1496 N PHE 519 31.904 125.886 100.761 1497 CA PHE 519 32.018 127.055 101.633 1498 C PHE 519 30.703 127.322 102.368 1499 O PHE 519 29.671 127.613 101.750 1500 CB PHE 519 32.373 128.276 100.787 1501 CG PHE 519 33.691 128.174 100.023 1502 CD1 PHE 519 34.897 128.234 100.711 1503 CD2 PHE 519 33.687 128.026 98.642 1504 CE1 PHE 519 36.097 128.144 100.018 1505 CE2 PHE 519 34.888 127.935 97.948 1506 CZ PHE 519 36.093 127.994 98.639 1507 N PHE 520 30.751 127.189 103.683 1508 CA PHE 520 29.621 127.572 104.543 1509 C PHE 520 29.669 129.073 104.799 1510 O PHE 520 30.418 129.565 105.656 1511 CB PHE 520 29.707 126.809 105.864 1512 CG PHE 520 28.732 127.276 106.945 1513 CD1 PHE 520 27.375 127.015 106.823 1514 CD2 PHE 520 29.207 127.969 108.052 1515 CE1 PHE 520 26.494 127.443 107.808 1516 CE2 PHE 520 28.327 128.398 109.036 1517 CZ PHE 520 26.968 128.136 108.913 1518 N PHE 521 28.886 129.782 104.010 1519 CA PHE 521 28.856 131.242 104.028 1520 C PHE 521 27.643 131.779 104.794 1521 O PHE 521 26.524 131.254 104.694 1522 CB PHE 521 28.790 131.631 102.557 1523 CG PHE 521 28.959 133.093 102.183 1524 CD1 PHE 521 27.862 133.943 102.151 1525 CD2 PHE 521 30.215 133.566 101.844 1526 CE1 PHE 521 28.028 135.268 101.780 1527 CE2 PHE 521 30.384 134.889 101.471 1528 CZ PHE 521 29.289 135.737 101.436 1529 N LYS 522 27.880 132.796 105.602 1530 CA LYS 522 26.775 133.467 106.291 1531 C LYS 522 26.363 134.716 105.519 1532 O LYS 522 27.034 135.757 105.564 1533 CB LYS 522 27.189 133.819 107.714 1534 CG LYS 522 26.037 134.439 108.497 1535 CD LYS 522 26.360 134.501 109.984 1536 CE LYS 522 26.529 133.096 110.553 1537 NZ LYS 522 26.904 133.135 111.975 1538 N GLY 523 25.191 134.610 104.910 1539 CA GLY 523 24.643 135.661 104.042 1540 C GLY 523 24.510 137.014 104.725 1541 O GLY 523 25.229 137.953 104.370 1542 N ASN 524 23.752 137.048 105.811 1543 CA ASN 524 23.474 138.304 106.527 1544 C ASN 524 24.560 138.723 107.538 1545 O ASN 524 24.251 139.461 108.483 1546 CB ASN 524 22.118 138.177 107.220 1547 CG ASN 524 21.257 139.421 106.982 1548 OD1 ASN 524 20.387 139.427 106.101 1549 ND2 ASN 524 21.517 140.459 107.760 1550 N ALA 525 25.773 138.202 107.418 1551 CA ALA 525 26.881 138.746 108.210 1552 C ALA 525 27.177 140.123 107.639 1553 O ALA 525 27.022 140.311 106.427 1554 CB ALA 525 28.108 137.857 108.065 1555 N TYR 526 27.736 141.025 108.428 1556 CA TYR 526 27.888 142.412 107.951 1557 C TYR 526 29.165 142.692 107.137 1558 O TYR 526 29.775 143.761 107.250 1559 CB TYR 526 27.770 143.381 109.116 1560 CG TYR 526 27.278 144.737 108.624 1561 CD1 TYR 526 26.399 144.777 107.549 1562 CD2 TYR 526 27.703 145.914 109.225 1563 CE1 TYR 526 25.943 145.996 107.069 1564 CE2 TYR 526 27.246 147.136 108.745 1565 CZ TYR 526 26.370 147.172 107.667 1566 OH TYR 526 25.935 148.383 107.176 1567 N TRP 527 29.546 141.711 106.336 1568 CA TRP 527 30.623 141.815 105.351 1569 C TRP 527 30.623 140.518 104.552 1570 O TRP 527 31.480 140.316 103.683 1571 CB TRP 527 31.987 142.089 105.990 1572 CG TRP 527 32.631 140.970 106.785 1573 CD1 TRP 527 33.499 140.020 106.297 1574 CD2 TRP 527 32.490 140.708 108.199 1575 NE1 TRP 527 33.868 139.207 107.319 1576 CE2 TRP 527 33.288 139.583 108.474 1577 CE3 TRP 527 31.776 141.323 109.217 1578 CZ2 TRP 527 33.349 139.079 109.764 1579 CZ3 TRP 527 31.846 140.816 110.508 1580 CH2 TRP 527 32.628 139.697 110.780 1581 N LYS 528 29.569 139.737 104.770 1582 CA LYS 528 29.373 138.418 104.141 1583 C LYS 528 30.609 137.514 104.206 1584 O LYS 528 31.523 137.630 103.383 1585 CB LYS 528 28.962 138.658 102.696 1586 CG LYS 528 27.580 139.285 102.592 1587 CD LYS 528 27.140 139.346 101.136 1588 CE LYS 528 25.725 139.891 100.987 1589 NZ LYS 528 25.328 139.923 99.569 1590 N VAL 529 30.580 136.541 105.100 1591 CA VAL 529 31.801 135.752 105.351 1592 C VAL 529 31.618 134.231 105.252 1593 O VAL 529 30.574 133.686 105.633 1594 CB VAL 529 32.285 136.152 106.746 1595 CG1 VAL 529 31.206 135.940 107.799 1596 CG2 VAL 529 33.592 135.477 107.160 1597 N VAL 530 32.580 133.578 104.615 1598 CA VAL 530 32.684 132.111 104.688 1599 C VAL 530 33.283 131.727 106.040 1600 O VAL 530 34.468 131.979 106.287 1601 CB VAL 530 33.630 131.605 103.601 1602 CG1 VAL 530 33.799 130.091 103.677 1603 CG2 VAL 530 33.183 132.021 102.208 1604 N ASN 531 32.505 131.048 106.864 1605 CA ASN 531 32.978 130.699 108.204 1606 C ASN 531 33.580 129.303 108.252 1607 O ASN 531 34.527 129.050 109.008 1608 CB ASN 531 31.796 130.714 109.164 1609 CG ASN 531 31.086 132.057 109.139 1610 OD1 ASN 531 31.714 133.115 109.018 1611 ND2 ASN 531 29.774 131.998 109.284 1612 N ASP 532 33.047 128.392 107.458 1613 CA ASP 532 33.536 127.004 107.547 1614 C ASP 532 33.738 126.338 106.189 1615 O ASP 532 32.805 126.214 105.389 1616 CB ASP 532 32.554 126.157 108.361 1617 CG ASP 532 32.491 126.578 109.832 1618 OD1 ASP 532 33.049 125.860 110.648 1619 OD2 ASP 532 31.705 127.473 110.120 1620 N LYS 533 34.956 125.887 105.953 1621 CA LYS 533 35.237 125.071 104.765 1622 C LYS 533 35.063 123.595 105.120 1623 O LYS 533 35.628 123.115 106.110 1624 CB LYS 533 36.659 125.359 104.303 1625 CG LYS 533 36.826 126.838 103.977 1626 CD LYS 533 38.251 127.163 103.546 1627 CE LYS 533 38.401 128.641 103.205 1628 NZ LYS 533 39.778 128.946 102.781 1629 N ASP 534 34.229 122.904 104.363 1630 CA ASP 534 33.881 121.518 104.718 1631 C ASP 534 33.693 120.652 103.463 1632 O ASP 534 33.887 121.125 102.334 1633 CB ASP 534 32.582 121.614 105.536 1634 CG ASP 534 32.301 120.389 106.411 1635 OD1 ASP 534 31.190 120.290 106.911 1636 OD2 ASP 534 33.225 119.613 106.623 1637 N LYS 535 33.518 119.358 103.675 1638 CA LYS 535 33.035 118.457 102.625 1639 C LYS 535 31.624 118.890 102.239 1640 O LYS 535 30.884 119.405 103.082 1641 CB LYS 535 32.943 117.052 103.210 1642 CG LYS 535 34.213 116.634 103.939 1643 CD LYS 535 34.032 115.266 104.590 1644 CE LYS 535 35.280 114.836 105.354 1645 NZ LYS 535 36.441 114.742 104.456 1646 N GLN 536 31.253 118.695 100.986 1647 CA GLN 536 29.896 119.064 100.558 1648 C GLN 536 28.925 117.933 100.834 1649 O GLN 536 28.578 117.615 101.977 1650 CB GLN 536 29.888 119.235 99.056 1651 CG GLN 536 31.093 119.991 98.544 1652 CD GLN 536 31.166 119.697 97.061 1653 OE1 GLN 536 30.430 118.836 96.565 1654 NE2 GLN 536 32.019 120.425 96.377 1655 N GLN 537 28.698 117.190 99.765 1656 CA GLN 537 27.824 116.009 99.767 1657 C GLN 537 28.342 114.842 100.606 1658 O GLN 537 27.525 114.156 101.231 1659 CB GLN 537 27.649 115.567 98.316 1660 CG GLN 537 28.736 116.100 97.379 1661 CD GLN 537 30.065 115.347 97.470 1662 OE1 GLN 537 30.109 114.151 97.782 1663 NE2 GLN 537 31.131 116.059 97.153 1664 N ASN 538 29.635 114.848 100.885 1665 CA ASN 538 30.245 113.842 101.749 1666 C ASN 538 29.959 114.114 103.231 1667 O ASN 538 30.115 113.212 104.061 1668 CB ASN 538 31.747 113.906 101.496 1669 CG ASN 538 32.408 112.577 101.827 1670 OD1 ASN 538 32.563 112.203 102.996 1671 ND2 ASN 538 32.841 111.906 100.774 1672 N SER 539 29.490 115.312 103.553 1673 CA SER 539 29.096 115.619 104.927 1674 C SER 539 27.572 115.693 105.052 1675 O SER 539 27.040 115.586 106.162 1676 CB SER 539 29.711 116.949 105.350 1677 OG SER 539 29.014 118.007 104.707 1678 N TRP 540 26.884 115.789 103.922 1679 CA TRP 540 25.413 115.787 103.932 1680 C TRP 540 24.864 114.368 103.946 1681 O TRP 540 23.709 114.137 104.334 1682 CB TRP 540 24.889 116.555 102.725 1683 CG TRP 540 25.103 118.050 102.846 1684 CD1 TRP 540 25.746 118.871 101.948 1685 CD2 TRP 540 24.664 118.894 103.932 1686 NE1 TRP 540 25.733 120.138 102.435 1687 CE2 TRP 540 25.098 120.195 103.622 1688 CE3 TRP 540 23.969 118.653 105.108 1689 CZ2 TRP 540 24.836 121.236 104.499 1690 CZ3 TRP 540 23.707 119.702 105.979 1691 CH2 TRP 540 24.141 120.989 105.677 1692 N LEU 541 25.689 113.443 103.492 1693 CA LEU 541 25.507 112.028 103.815 1694 C LEU 541 26.897 111.498 104.135 1695 O LEU 541 27.644 111.122 103.223 1696 CB LEU 541 24.891 111.289 102.633 1697 CG LEU 541 24.109 110.037 103.041 1698 CD1 LEU 541 24.995 108.878 103.483 1699 CD2 LEU 541 23.055 110.363 104.094 1700 N PRO 542 27.220 111.488 105.420 1701 CA PRO 542 28.606 111.335 105.872 1702 C PRO 542 29.199 109.996 105.465 1703 O PRO 542 28.696 108.932 105.858 1704 CB PRO 542 28.562 111.483 107.362 1705 CG PRO 542 27.135 111.770 107.796 1706 CD PRO 542 26.308 111.807 106.524 1707 N ALA 543 30.259 110.062 104.674 1708 CA ALA 543 30.923 108.837 104.229 1709 C ALA 543 31.562 108.148 105.420 1710 O ALA 543 32.441 108.695 106.097 1711 CB ALA 543 31.974 109.161 103.181 1712 N ASN 544 31.073 106.952 105.687 1713 CA ASN 544 31.501 106.212 106.876 1714 C ASN 544 32.966 105.822 106.740 1715 O ASN 544 33.393 105.260 105.727 1716 CB ASN 544 30.603 104.991 107.066 1717 CG ASN 544 29.449 105.256 108.047 1718 OD1 ASN 544 29.261 104.474 108.984 1719 ND2 ASN 544 28.702 106.333 107.851 

1. An isolated nucleic acid molecule comprising a polynucleotide sequence selected from the group consisting of: (a) an isolated polynucleotide encoding a polypeptide comprising amino acids 1 to 569 of SEQ ID NO:2; (b) an isolated polynucleotide encoding a polypeptide comprising amino acids 2 to 569 of SEQ ID NO:2; and (c) an isolated polynucleotide encoding a mature polypeptide comprising amino acids 25 to 569 of SEQ ID NO:2, wherein the encoded polypeptide has metalloproteinase activity.
 2. The isolated nucleic acid molecule of claim 1, wherein said polynucleotide is (a).
 3. The isolated nucleic acid molecule of claim 2, wherein said polynucleotide comprises nucleotides 1 to 1707 of SEQ ID NO:1.
 4. The isolated nucleic acid molecule of claim 1, wherein said polynucleotide is (b).
 5. The isolated nucleic acid molecule of claim 4, wherein said polynucleotide comprises nucleotides 4 to 1707 of SEQ ID NO:1.
 6. The isolated nucleic acid molecule of claim 1, wherein said polynucleotide is (c).
 7. The isolated nucleic acid molecule of claim 6, wherein said polynucleotide comprises nucleotides 73 to 1707 of SEQ ID NO:1.
 8. An isolated polynucleotide comprising the complete complementary sequence of(a), (b), or (c) of claim
 1. 9. The isolated polynucleotide according to claim 1 wherein said polynucleotide encodes a polypeptide containing a single amino acid substitution.
 10. A recombinant vector comprising the isolated nucleic acid molecule of claim
 1. 11. A recombinant host cell comprising the vector sequence of claim
 10. 12. A method of making an isolated polypeptide comprising: (a) culturing the recombinant host cell of claim 11 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide.
 13. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence that is at least 99.9% identical to nucleotides 4 to 1707 of SEQ ID NO:1, wherein percent identity is calculated using a CLUSTALW global sequence alignment using default parameters, and wherein said polynucleotide encodes a polypeptide that has metalloproteinase activity.
 14. An isolated nucleic acid molecule comprising a polynucleotide that encodes a polypeptide having a polypeptide sequence that is at least 99.7% identical to amino acids 2 to 569 of SEQ ID NO:2, wherein percent identity is calculated using a CLUSTALW global sequence alignment, and wherein said polypeptide has metalloproteinase activity.
 15. The isolated polynucleotide according to claim 14 wherein said polynucleotide contains a single nucleotide substitution. 